Simulated Moving Bed Chromatographic Separation Process

ABSTRACT

The present invention provides a chromatographic separation process for recovering a polyunsaturated fatty acid (PUFA) product, from a feed mixture, which process comprises introducing the feed mixture to a simulated or actual moving bed chromatography apparatus having a plurality of linked chromatography columns containing, as eluent, an aqueous alcohol, wherein the apparatus has a plurality of zones comprising at least a first zone and a second zone, each zone having an extract stream and a raffinate stream from which liquid can be collected from said plurality of linked chromatography columns, and wherein (a) a raffinate stream containing the PUFA product together with more polar components is collected from a column in the first zone and introduced to a nonadjacent column in the second zone, and/or (b) an extract stream containing the PUFA product together with less polar components is collected from a column in the second zone and introduced to a nonadjacent column in the first zone, said PUFA product being separated from different components of the feed mixture in each zone.

The present invention relates to an improved chromatographicfractionation process for purifying polyunsaturated fatty acids (PUFAs)and derivatives thereof. In particular, the present invention relates toan improved simulated or actual moving bed chromatographic separationprocess for purifying PUFAs and derivatives thereof.

Fatty acids, in particular PUFAs, and their derivatives are precursorsfor biologically important molecules, which play an important role inthe regulation of biological functions such as platelet aggregation,inflammation and immunological responses. Thus, PUFAs and theirderivatives may be therapeutically useful in treating a wide range ofpathological conditions including CNS conditions; neuropathies,including diabetic neuropathy; cardiovascular diseases; general immunesystem and inflammatory conditions, including inflammatory skindiseases.

PUFAs are found in natural raw materials, such as vegetable oils andmarine oils. Such PUFAs are however, frequently present in such oils inadmixture with saturated fatty acids and numerous other impurities.PUFAs should therefore desirably be purified before nutritional orpharmaceutical uses.

Unfortunately, PUFAs are extremely fragile. Thus, when heated in thepresence of oxygen, they are prone to isomerization, peroxidation andoligomerization. The fractionation and purification of PUFA products toprepare pure fatty acids is therefore difficult. Distillation, evenunder vacuum, can lead to non-acceptable product degradation.

Simulated and actual moving bed chromatography are known techniques,familiar to those of skill in the art. The principle of operationinvolves countercurrent movement of a liquid eluent phase and a solidadsorbent phase. This operation allows minimal usage of solvent makingthe process economically viable. Such separation technology has foundseveral applications in diverse areas, including hydrocarbons,industrial chemicals, oils, sugars and APIs. Such separation technologyhas also been applied to purify PUFAs and their derivatives.

As is well known, in a conventional stationary bed chromatographicsystem, a mixture whose components are to be separated percolatesthrough a container. The container is generally cylindrical, and istypically referred to as the column. The column contains a packing of aporous material (generally called the stationary phase) exhibiting ahigh permeability to fluids. The percolation velocity of each componentof the mixture depends on the physical properties of that component sothat the components exit from the column successively and selectively.Thus, some of the components tend to fix strongly to the stationaryphase and thus will percolate slowly, whereas others tend to fix weaklyand exit from the column more quickly. Many different stationary bedchromatographic systems have been proposed and are used for bothanalytical and industrial production purposes.

In contrast, a simulated moving bed system consists of a number ofindividual columns containing adsorbent which are connected together inseries. Eluent is passed through the columns in a first direction. Theinjection points of the feedstock and the eluent, and the separatedcomponent collection points in the system, are periodically shifted bymeans of a series of valves. The overall effect is to simulate theoperation of a single column containing a moving bed of the solidadsorbent. Thus, a simulated moving bed system consists of columnswhich, as in a conventional stationary bed system, contain stationarybeds of solid adsorbent through which eluent is passed, but in asimulated moving bed system the operation is such as to simulate acontinuous countercurrent moving bed.

Processes and equipment for simulated moving bed chromatography aredescribed in several patents, including U.S. Pat. No. 2,985,589, U.S.Pat. No. 3,696,107, U.S. Pat. No. 3,706,812, U.S. Pat. No. 3,761,533,FR-A-2103302, FR-A-2651148 and FR-A-2651149, the entirety of which areincorporated herein by reference. The topic is also dealt with at lengthin “Preparative and Production Scale Chromatography”, edited by Ganetsosand Barker, Marcel Dekker Inc, New York, 1993, the entirety of which isincorporated herein by reference.

An actual moving bed system is similar in operation to a simulatedmoving bed system. However, rather than shifting the injection points ofthe feed mixture and the eluent, and the separated component collectionpoints by means of a system of valves, instead a series of adsorptionunits (i.e. columns) are physically moved relative to the feed anddrawoff points. Again, operation is such as to simulate a continuouscountercurrent moving bed.

Processes and equipment for actual moving bed chromatography aredescribed in several patents, including U.S. Pat. No. 6,979,402, U.S.Pat. No. 5,069,883 and U.S. Pat. No. 4,764,276, the entirety of whichare incorporated herein by reference.

Simulated and actual moving bed technology is generally only suitablefor separating binary mixtures. Thus, a more polar component will movewith the eluent, and be collected as a raffinate stream, and a lesspolar component will move with the adsorbent, and be collected as anextract stream. It is therefore difficult to use simulated or actualmoving bed technology to separate a desired product from a crude mixturecontaining both polar and non-polar impurities. This limits theapplicability of such techniques in purifying PUFA products from fishoils, for example.

Accordingly, when simulated or actual moving bed technology has beenused to separate PUFAs from natural oils in the past, it is generallynecessary first to subject the natural oil to a preliminary separationstep (e.g. fixed column chromatography) before purifying theintermediate product obtained using simulated or actual moving bedtechnology (see, for example, EP-A-0697034). Typically, the initialpurification step removes polar or non-polar components, thus creatingan essentially binary mixture which is then subjected to moving bedchromatography.

This process of separating a binary mixture is illustrated withreference to FIG. 1. The concept of a simulated or actual continuouscountercurrent chromatographic separation process is explained byconsidering a vertical chromatographic column containing stationaryphase S divided into sections, more precisely into four superimposedsub-zones I, II, III and IV going from the bottom to the top of thecolumn. The eluent is introduced at the bottom at IE by means of a pumpP. The mixture of the components A and B which are to be separated isintroduced at IA+B between sub-zone II and sub-zone III. An extractcontaining mainly B is collected at SB between sub-zone I and sub-zoneII, and a raffinate containing mainly A is collected at SA betweensub-zone III and sub-zone IV.

In the case of a simulated moving bed system, a simulated downwardmovement of the stationary phase S is caused by movement of theintroduction and collection points relative to the solid phase. In thecase of an actual moving bed system, downward movement of the stationaryphase S is caused by movement of the various chromatographic columnsrelative to the introduction and collection points. In FIG. 1, eluentflows upward and mixture A+B is injected between sub-zone II andsub-zone III. The components will move according to theirchromatographic interactions with the stationary phase, for exampleadsorption on a porous medium. The component B that exhibits strongeraffinity to the stationary phase (the slower running component) will bemore slowly entrained by the eluent and will follow it with delay. Thecomponent A that exhibits the weaker affinity to the stationary phase(the faster running component) will be easily entrained by the eluent.If the right set of parameters, especially the flow rate in each zone,are correctly estimated and controlled, the component A exhibiting theweaker affinity to the stationary phase will be collected betweensub-zone III and sub-zone IV as a raffinate and the component Bexhibiting the stronger affinity to the stationary phase will becollected between sub-zone I and sub-zone II as an extract.

It will therefore be appreciated that the conventional moving bed systemschematically illustrated in FIG. 1 is limited to binary fractionation.

Accordingly, there is a need for a single simulated or actual moving bedchromatographic separation process that can separate PUFAs or theirderivatives from both faster and slower running components (i.e. morepolar and less polar impurities), to produce an essentially pure PUFA orderivative thereof. It is further desirable that the process shouldinvolve inexpensive eluents which operate under standard temperature andpressure conditions.

It has now been surprisingly found that a PUFA product can beeffectively purified with a single simulated or actual moving bedapparatus using an aqueous alcohol eluent. The present inventiontherefore provides a chromatographic separation process for recovering apolyunsaturated fatty acid (PUFA) product, from a feed mixture, whichprocess comprises introducing the feed mixture to a simulated or actualmoving bed chromatography apparatus having a plurality of linkedchromatography columns containing, as eluent, an aqueous alcohol,wherein the apparatus has a plurality of zones comprising at least afirst zone and second zone, each zone having an extract stream and araffinate stream from which liquid can be collected from said pluralityof linked chromatography columns, and wherein (a) a raffinate streamcontaining the PUFA product together with more polar components iscollected from a column in the first zone and introduced to anonadjacent column in the second zone, and/or (b) an extract streamcontaining the PUFA product together with less polar components iscollected from a column in the second zone and introduced to anonadjacent column in the first zone, said PUFA product being separatedfrom different components of the feed mixture in each zone.

Also provided is a PUFA product obtainable by the process of the presentinvention.

The PUFA products produced by the process of the present invention areproduced in high yield, and have high purity. Further, the content ofthe distinctive impurities which typically arise from distillation ofPUFAs is very low. As used herein, the term “isomeric impurities” isused to denote those impurities typically produced during thedistillation of PUFA-containing natural oils. These include PUFAisomers, peroxidation and oligomerization products.

FIG. 1 illustrates the basic principles of a simulated or actual movingbed process for separating a binary mixture.

FIG. 2 illustrates a first preferred embodiment of the invention whichis suitable for separating EPA from faster and slower running components(i.e. more polar and less polar impurities).

FIG. 3 illustrates a second preferred embodiment of the invention whichis suitable for separating DHA from faster and slower running components(i.e. more polar and less polar impurities).

FIG. 4 illustrates in more detail the first preferred embodiment of theinvention which is suitable for separating EPA from faster and slowerrunning components (i.e. more polar and less polar impurities).

FIG. 5 illustrates in more detail the second preferred embodiment of theinvention which is suitable for separating DHA from faster and slowerrunning components (i.e. more polar and less polar impurities).

FIG. 6 illustrates in more detail an alternative method for the firstpreferred embodiment of the invention which is suitable for separatingEPA from faster and slower running components (i.e. more polar and lesspolar impurities).

FIG. 7 illustrates in more detail an alternative method for the secondpreferred embodiment of the invention which is suitable for separatingDHA from faster and slower running components (i.e. more polar and lesspolar impurities).

FIG. 8 illustrates a particularly preferred embodiment of the inventionfor purifying EPA from faster and slower running components (i.e. morepolar and less polar impurities).

FIG. 9 illustrates an alternative method for a particularly preferredembodiment of the invention for purifying EPA from faster and slowerrunning components (i.e. more polar and less polar impurities).

FIG. 10 illustrates a particularly preferred embodiment of the inventionfor purifying EPA from faster and slower running components (i.e. morepolar and less polar impurities).

FIG. 11 shows a GC analysis of an EPA product produced in accordancewith the invention.

FIG. 12 shows GC FAMES traces of first extract and raffinate streamsobtained in accordance with the invention.

FIG. 13 shows GC FAMES traces of second extract and raffinate streamsobtained in accordance with the invention.

FIG. 14 shows a GC FAMES trace of a DHA product produced in accordancewith the invention.

FIG. 15 shows a GC FAMES trace of a DHA product produced bydistillation.

The term “polyunsaturated fatty acid” (PUFA) refers to fatty acids thatcontain more than one double bond. Such PUFAs are well known to theperson skilled in the art. As used herein, a PUFA derivative is a PUFAin the form of a mono-, di- or tri-glyceride, ester, phospholipid,amide, lactone, or salt. Triglycerides and esters are preferred. Estersare more preferred. Esters are typically alkyl esters, preferably C₁-C₆alkyl esters, more preferably C₁-C₄ alkyl esters. Examples of estersinclude methyl and ethyl esters. Ethyl esters are most preferred.

As used herein, the term “PUFA product” refers to a product comprisingone or more polyunsaturated fatty acids (PUFAs), and/or derivativesthereof, typically of nutritional or pharmaceutical significance.Typically, the PUFA product is a single PUFA or derivative thereof.Alternatively, the PUFA product is a mixture of two or more PUFAs orderivatives thereof, for example two.

As used herein, the term “zone” refers to a plurality of linkedchromatography columns containing, as eluent, an aqueous alcohol, andhaving one or more injection points for a feed mixture stream, one ormore injection points for water and/or alcohol, a raffinate take-offstream from which liquid can be collected from said plurality of linkedchromatography columns, and an extract take-off stream from which liquidcan be collected from said plurality of linked chromatography columns.Typically, each zone has only one injection point for a feed mixture. Inone embodiment, each zone has only one injection point for the aqueousalcohol eluent. In another embodiment, each zone has two or moreinjection points for water and/or alcohol.

The term “raffinate” is well known to the person skilled in the art. Inthe context of actual and simulated moving bed chromatography it refersto the stream of components that move more rapidly with the liquideluent phase compared with the solid adsorbent phase. Thus, a raffinatestream is typically enriched with more polar components, and depleted ofless polar components compared with a feed stream.

The term “extract” is well known to the person skilled in the art. Inthe context of actual and simulated moving bed chromatography it refersto the stream of components that move more rapidly with the solidadsorbent phase compared with the liquid eluent phase. Thus, an extractstream is typically enriched with less polar components, and depleted ofmore polar components compared with a feed stream.

As used herein the term “nonadjacent” when applied to columns in thesame apparatus refers to columns separated by one or more columns,preferably 3 or more columns, 15 more preferably 5 or more columns, mostpreferably about 5 columns.

Thus, where (a) a raffinate stream containing the PUFA product togetherwith more polar components is collected from a column in the first zoneand introduced to a nonadjacent column in the second zone, the raffinatestream collected from the first zone is the feed mixture for the secondzone. Where (b) an extract stream containing the PUFA product togetherwith less polar components is collected from a column in the second zoneand introduced to a nonadjacent column in the first zone, the extractstream collected from the second zone is the feed mixture in the firstzone.

Typically, the PUFA product comprises at least one ω-3 or ω-6 PUFA,preferably at least one ω-3 PUFA. Examples of ω-3 PUFAs includealpha-linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid(ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA),docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA). SDA, EPA,DPA and DHA are preferred. EPA and DHA are more preferred. Examples ofω-6 PUFAs include linoleic acid (LA), gamma-linolenic acid (GLA),eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), arachidonic acid(ARA), docosadienoic acid, adrenic acid and docosapentaenoic (ω-6) acid.LA, ARA, GLA and DGLA are preferred.

In one embodiment, the PUFA product is EPA and/or EPA ethyl ester (EE)

In another embodiment, the PUFA product is DHA and/or EPA ethyl ester(EE).

In a yet further embodiment, the PUFA product is a mixture of EPA andDHA and/or EPA EE and DHA EE.

Suitable feed mixtures for fractionating by the process of the presentinvention may be obtained from natural sources including vegetable andanimal oils and fats, and from synthetic sources including oils obtainedfrom genetically modified plants, animals and micro organisms includingyeasts. Examples include fish oils, algal and microalgal oils and plantoils, for example borage oil, Echium oil and evening primrose oil. Inone embodiment, the feed mixture is a fish oil. In another embodiment,the feed mixture is an algal oil. Algal oils are particularly suitablewhen the desired PUFA product is EPA and/or DHA. Genetically modifiedSafflower oil is particularly suitable when the desired PUFA product isGLA. Genetically modified yeast is particularly suitable when thedesired PUFA product is EPA.

The feed mixture may undergo chemical treatment before fractionation bythe process of the invention. For example, it may undergo glyceridetransesterification or glyceride hydrolysis followed in certain cases byselective processes such as crystallisation, molecular distillation,urea fractionation, extraction with silver nitrate or other metal saltsolutions, iodolactonisation or supercritical fluid fractionation.

The feed mixtures typically contain the PUFA product and at least onemore polar component and at least one less polar component. The lesspolar components have a stronger adherence to the adsorbent used in theprocess of the present invention than does the PUFA product. Duringoperation, such less polar components typically move with the solidadsorbent phase in preference to the liquid eluent phase. The more polarcomponents have a weaker adherence to the adsorbent used in the processof the present invention than does the PUFA product. During operation,such more polar components typically move with the liquid eluent phasein preference to the solid adsorbent phase. In general, more polarcomponents will be separated into a raffinate stream, and less polarcomponents will be separated into an extract stream.

Examples of the more and less polar components include (1) othercompounds occurring in natural oils (e.g. marine oils or vegetableoils), (2) byproducts formed during storage, refining and previousconcentration steps and (3) contaminants from solvents or reagents whichare utilized during previous concentration or purification steps.

Examples of (1) include other unwanted PUFAs; saturated fatty acids;sterols, for example cholesterol; vitamins; and environmentalpollutants, such as polychlorobiphenyl (PCB), polyaromatic hydrocarbon(PAH) pesticides, chlorinated pesticides, dioxines and heavy metals.PCB, PAH, dioxines and chlorinated pesticides are all highly non-polarcomponents.

Examples of (2) include isomers and oxidation or decomposition productsfrom the PUFA product, for instance, auto-oxidation polymeric productsof fatty acids or their derivatives.

Examples of (3) include urea which may be added to remove saturated ormono-unsaturated fatty acids from the feed mixture.

Preferably, the feed mixture is a PUFA-containing marine oil, morepreferably a marine oil comprising EPA and/or DHA.

A typical feed mixture for preparing concentrated EPA by the process ofthe present invention comprises 50-75% EPA, 0 to 10% DHA, and othercomponents including other essential ω-3 and ω-6 fatty acids.

A preferred feed mixture for preparing concentrated EPA by the processof the present invention comprises 55% EPA, 5% DHA, and other componentsincluding other essential ω-3 and ω-6 fatty acids. DHA is less polarthan EPA.

A typical feed mixture for preparing concentrated DHA by the process ofthe present invention comprises 50-75% DHA, 0 to 10% EPA, and othercomponents including other essential ω-3 and ω-6 fatty acids.

A preferred feed mixture for preparing concentrated DHA by the processof the present invention comprises 75% DHA, 7% EPA and other componentsincluding other essential ω-3 and ω-6 fatty acids. EPA is more polarthan DHA.

A typical feed mixture for preparing a concentrated mixture of EPA andDHA by the process of the present invention comprises greater than 33%EPA, and greater than 22% DHA.

The process of the invention requires a plurality of zones in saidchromatography apparatus. Typically, two or more zones are used. Thenumber of zones is not particularly limited, but in general there are 2to 5 zones. Preferably, there are two or three zones, more preferablythere are two zones.

Typically, the components separated in each zone of the apparatus usedin the process of the present invention have different polarities.

Typically, a) the aqueous alcohol eluent present in each zone has adifferent water:alcohol ratio; and/or

-   -   (b) the rate at which liquid collected via the extract and        raffinate streams in each zone is recycled back into the same        zone is adjusted such that the PUFA product can be separated        from different components of the feed mixture in each zone.

When the apparatus used in the process of the present invention has twozones, the present invention typically provides a chromatographicseparation process for recovering a polyunsaturated fatty acid (PUFA)product, from a feed mixture, which process comprises introducing thefeed mixture to a simulated or actual moving bed chromatographyapparatus having a plurality of linked chromatography columnscontaining, as eluent, an aqueous alcohol, wherein the apparatus has afirst zone and a second zone, each zone having an extract stream and araffinate stream from which liquid can be collected from said pluralityof linked chromatography columns, and wherein (a) a raffinate streamcontaining the PUFA product together with more polar components iscollected from a column in the first zone and introduced to anonadjacent column in the second zone, and/or (b) an extract streamcontaining the PUFA product together with less polar components iscollected from a column in the second zone and introduced to anonadjacent column in the first zone, said PUFA product being separatedfrom less polar components of the feed mixture in the first zone, andsaid PUFA product being separated from more polar components of the feedmixture in the second zone.

Typically, when the apparatus used in the process of the presentinvention contains two zones, the eluent in the first zone contains morealcohol than the eluent in the second zone, and the second zone isdownstream of the first zone with respect to the flow of eluent in thesystem. Thus, the eluent in the system typically moves from the firstzone to the second zone. Conversely, the solid adsorbent phase typicallymoves from the second zone to the first zone. Typically, the two zonesdo not overlap, i.e. there are no chromatographic columns which are inboth zones.

In a further embodiment of the invention, the apparatus has a firstzone, a second zone and a third zone. The water:alcohol ratios of theaqueous alcohol eluent present in the first, second and third zones aretypically different. As will be evident to one skilled in the art, thishas the consequence that impurities having different polarities can beremoved in each zone.

Preferably, when the apparatus has three zones, the eluent in the firstzone contains more alcohol than the eluent in the second zone and thethird zone and the first zone is upstream of the second and third zoneswith respect to the flow of eluent in the system. Typically, the eluentin the second zone contains more alcohol than the eluent in the thirdzone and the second zone is upstream of the third zone with respect tothe flow of eluent in the system. Typically, in the first zone, saidPUFA product is separated from components of the feed mixture which areless polar than the PUFA product. Typically, in the second zone, saidPUFA product is separated from components of the feed mixture which areless polar than the PUFA product but more polar than the componentsseparated in the first zone. Typically, in the third zone, said PUFAproduct is separated from components of the feed mixture which are morepolar than the PUFA product.

In a further embodiment, in the first zone, said PUFA product isseparated from components of the feed mixture which are less polar thanthe PUFA product, in the second zone, said PUFA product is separatedfrom components of the feed mixture which are more polar than the PUFAproduct, and in the third zone, said PUFA product is separated fromcomponents of the feed mixture which are more polar than the PUFAproduct and also more polar than, the components separated in the secondzone.

Such a setup having three zones would be suitable for separating EPA andDHA from a mixture containing impurities which are less polar than DHAand EPA and also containing impurities which are more polar than EPA. Inthe first zone, the components which are less polar than DHA and EPA areremoved as an extract stream and a raffinate stream comprising DHA, EPAand components which are more polar than EPA is collected and introducedinto the second zone. In the second zone, DHA is removed as an extractstream and a raffinate stream comprising EPA and components which aremore polar than EPA is collected and introduced into the third zone. Inthe third zone, the components which are more polar than EPA are removedas a raffinate stream and purified EPA is collected as an extractstream. In this embodiment, the purified EPA is the purified PUFAproduct. Such a setup has an advantage that a secondary PUFA may also berecovered. In this case, the secondary PUFA is the DHA collected as theextract stream from the second zone.

Typically, in addition to said PUFA product, an additional secondaryPUFA product is collected in the chromatographic separation process ofthe invention. Preferably, the PUFA product is EPA and the additionalsecondary PUFA product is DHA.

In a further embodiment of the invention, the apparatus is configured tocollect a PUFA product which is a concentrated mixture of EPA and DHA.Thus, a feed mixture is used which contains EPA, DHA, components whichare more polar than EPA and DHA, and components which are less polarthan EPA and DHA. In the first zone, less polar material than EPA andDHA is removed. In the second zone, material which is more polar thanEPA and DHA is removed, and a concentrated mixture of EPA and DHA iscollected as the PUFA product.

Any known simulated or actual moving bed chromatography apparatus may beutilised for the purposes of the method of the present invention, aslong as the apparatus is configured with the multiple, in particulartwo, zones which characterise the process of the present invention.Those apparatuses described in U.S. Pat. No. 2,985,589, U.S. Pat. No.3,696,107, U.S. Pat. No. 3,706,812, U.S. Pat. No. 3,761,533,FR-A-2103302, FR-A-2651148, FR-A-2651149, U.S. Pat. No. 6,979,402, U.S.Pat. No. 5,069,883 and U.S. Pat. No. 4,764,276 may all be used ifconfigured in accordance with the process of the present invention.

The number of columns used in the apparatus is not particularly limited.A skilled person would easily be able to determine an appropriate numberof columns to use. The number of columns is typically 8 or more,preferably 15 or more. In a more preferred embodiment 15 or 16 columnsare used. In another more preferred embodiment, 19 or 20 columns areused. In other more preferred embodiments, 30 or more columns are used.Typically, there are no more than 50 columns, preferably no more than40.

Each zone typically consists of an approximately equal share of thetotal number of columns. Thus, in the case of an apparatus configuredwith two zones, each zone typically consists of approximately half ofthe total number of chromatographic columns in the system. Thus, thefirst zone typically comprises 4 or more, preferably 8 or more, morepreferably about 8 columns. The second zone typically comprises 4 ormore, preferably 7 or more, more preferably 7 or 8 columns.

The dimensions of the columns used in the apparatus are not particularlylimited, and will depend on the volume of feed mixture to be purified. Askilled person would easily be able to determine appropriately sizedcolumns to use. The diameter of each column is typically between 10 and500 mm, preferable between 25 and 250 mm, more preferable between 50 and100 mm, and most preferably between 70 and 80 mm. The length of eachcolumn is typically between 10 and 200 cm, preferably between 25 and 150cm, more preferably between 70 and 110 cm, and most preferably between80 and 100 cm.

The columns in each zone typically have identical dimensions but may,for certain applications, have different dimensions.

The flow rates to the column are limited by maximum pressures across theseries of columns and will depend on the column dimensions and particlesize of the solid phases. One skilled in the art will easily be able toestablish the required flow rate for each column dimension to ensureefficient desorption. Larger diameter columns will in general needhigher flows to maintain linear flow through the columns.

For the typical column sizes outlined above, and for an apparatus havingtwo zones, typically the flow rate of eluent into the first zone is from1 to 4.5 L/min, preferably from 1.5 to 2.5 L/min. Typically, the flowrate of the extract from the first zone is from 0.1 to 2.5 L/min,preferably from 0.5 to 2.25 L/min. In embodiments where part of theextract from the first zone is recycled back into the first zone, theflow rate of recycle is typically from 0.7 to 1.4 L/min, preferablyabout 1 L/min. Typically, the flow rate of the raffinate from the firstzone is from 0.2 to 2.5 L/min, preferably from 0.3 to 2.0 L/min. Inembodiments where part of the raffinate from the first zone is recycledback into the first zone, the flow rate of recycle is typically from 0.3to 1.0 L/min, preferably about 0.5 L/min. Typically, the flow rate ofintroduction of the feed mixture into the first zone is from 5 to 150mL/min, preferably from 10 to 100 mL/min, more preferably from 20 to 60mL/min.

For the typical column sizes outlined above, and for an apparatus havingtwo zones, typically the flow rate of eluent into the second zone isfrom 1 to 4 L/min, preferably from 1.5 to 3.5 L/min. Typically, the flowrate of the extract from the second zone is from 0.5 to 2 L/min,preferably from 0.7 to 1.9 L/min. In embodiments where part of theextract from the second zone is recycled back into the second zone, theflow rate of recycle is typically from 0.6 to 1.4 L/min, preferably from0.7 to 1.1 L/min, more preferably about 0.9 L/min. Typically, the flowrate of the raffinate from the second zone is from 0.5 to 2.5 L/min,preferably from 0.7 to 1.8 L/min, more preferably about 1.4 L/min.

As the skilled person will appreciate, references to rates at whichliquid is collected or removed via the various extract and raffinatestreams refer to volumes of liquid removed in an amount of time,typically L/minute. Similarly, references to rates at which liquid isrecycled back into the same zone, typically to an adjacent column in thesame zone, refer to volumes of liquid recycled in an amount of time,typically L/minute.

Typically, part of one or more of the extract stream from the firstzone, the raffinate stream from the first zone, the extract stream fromthe second zone, and the raffinate stream from the second zone arerecycled back into the same zone, typically into an adjacent column inthe same zone.

This recycle is different from the feeding of an extract or raffinatestream into a nonadjacent column in another zone. Rather, the recycleinvolves feeding part of the extract or raffinate stream out of a zoneback into the same zone, typically into an adjacent column in the samezone.

The rate at which liquid collected via the extract or raffinate streamfrom the first or second zones is recycled back into the same zone isthe rate at which liquid collected via that stream is fed back into thesame zone, typically into an adjacent column in the same zone. This canbe seen with reference to FIG. 9. The rate of recycle of extract in thefirst zone is the rate at which extract collected from the bottom ofcolumn 2 is fed into the top of column 3, i.e. the flow rate of liquidinto the top of column 3. The rate of recycle of extract in the secondzone is the rate at which extract collected at the bottom of column 10is fed into the top of column 11, i.e. the flow rate of liquid into thetop of column 11.

Recycle of the extract and/or raffinate streams is typically effected byfeeding the liquid collected via that stream into a container, and thenpumping an amount of that liquid from the container back into the samezone. In this case, the rate of recycle of liquid collected via aparticular extract or raffinate stream, typically back into an adjacentcolumn in the same zone, is the rate at which liquid is pumped out ofthe container back into the same zone, typically into an adjacentcolumn.

As the skilled person will appreciate, the amount of liquid beingintroduced into a zone via the eluent and feedstock streams is balancedwith the amount of liquid removed from a zone, and recycled back intothe same zone. Thus, with reference to FIG. 9, for the extract stream,the flow rate of eluent (desorbent) into the first or second zone (D) isequal to the rate at which liquid collected via the extract stream fromthat zone accumulates in a container (E1/E2) added to the rate at whichextract is recycled back into the same zone (D−E1/D−E2). For theraffinate stream in a zone, the rate at which extract is recycled backinto a zone (D−E1/D−E2) added to the rate at which feedstock isintroduced into a zone (F/R1) is equal to the rate at which liquidcollected via the raffinate stream from that zone accumulates in acontainer (R1/R2) added to the rate at which raffinate is recycled backinto the same zone (D+F−E1−R1/D+R1−E2−R2).

The rate at which liquid collected from a particular extract orraffinate stream from a zone accumulates in a container can also bethought of as the net rate of removal of that extract or raffinatestream from that zone.

Typically, the rate at which liquid collected via the extract stream outof the first zone is recycled back into the first zone differs from therate at which liquid collected via the extract stream out of the secondzone is recycled back into the second zone, and/or the rate at whichliquid collected via the raffinate stream out of the first zone isrecycled back into the first zone differs from the rate at which liquidcollected via the raffinate stream out of the second zone is recycledback into the second zone.

Varying the rate at which liquid collected via the extract and/orraffinate streams in each zone is recycled back into the same zone hasthe effect of varying the amount of more polar and less polar componentspresent in the other extract and raffinate streams. Thus, for example, alower extract recycle rate results in fewer of the less polar componentsin that zone being carried through to the raffinate stream in that zone.A higher extract recycle rate results in more of the less polarcomponents in that zone being carried through to the raffinate stream inthat zone. This can be seen, for example, in the specific embodiment ofthe invention shown in FIG. 6. The rate at which liquid collected viathe extract stream in the first zone is recycled back into the same zone(D−E1) will affect to what extent any of component A is carried throughto the raffinate stream in the first zone (R1).

Typically, the rate at which liquid collected via the extract streamfrom the first zone is recycled back into the first zone is faster thanthe rate at which liquid collected via the extract stream from thesecond zone is recycled back into the second zone. Preferably, araffinate stream containing the PUFA product together with more polarcomponents is collected from a column in the first zone and introducedto a nonadjacent column in the second zone, and the rate at which liquidcollected via the extract stream from the first zone is recycled backinto the first zone is faster than the rate at which liquid collectedvia the extract stream from the second zone is recycled back into thesecond zone.

Alternatively, the rate at which liquid collected via the extract streamfrom the first zone is recycled back into the first zone is slower thanthe rate at which liquid collected via the extract stream from thesecond zone is recycled back into the second zone.

Typically, the rate at which liquid collected via the raffinate streamfrom the second zone is recycled back into the second zone is fasterthan the rate at which liquid collected via the raffinate stream fromthe first zone is recycled back into the first zone. Preferably, anextract stream containing the PUFA product together with less polarcomponents is collected from a column in the second zone and introducedto a nonadjacent column in the first zone, and the rate at which liquidcollected via the raffinate stream from the second zone is recycled backinto the second zone is faster than the rate at which liquid collectedvia the raffinate stream from the first zone is recycled back into thefirst zone.

Alternatively, the rate at which liquid collected via the raffinatestream from the second zone is recycled back into the second zone isslower than the rate at which liquid collected via the raffinate streamfrom the first zone is recycled back into the first zone.

The step time, i.e. the time between shifting the points of injection ofthe feed mixture and eluent, and the various take off points of thecollected fractions, is not particularly limited, and will depend on thenumber and dimensions of the columns used, and the flow rate through theapparatus. A skilled person would easily be able to determineappropriate step times to use in the process of the present invention.The step time is typically from 100 to 1000 seconds, preferably from 200to 800 seconds, more preferably from about 250 to about 750 seconds. Insome embodiments, a step time of from 100 to 400 seconds, preferably 200to 300 seconds, more preferably about 250 seconds, is appropriate. Inother embodiments, a step time of from 600 to 900 seconds, preferably700 to 800 seconds, more preferably about 750 seconds is appropriate.

In the process of the present invention, actual moving bedchromatography is preferred.

Conventional adsorbents known in the art for actual and simulated movingbed systems may be used in the process of the present invention. Eachchromatographic column may contain the same or a different adsorbent.Typically, each column contains the same adsorbent. Examples of suchcommonly used materials are polymeric beads, preferably polystyrenereticulated with DVB (divinylbenzene); and silica gel, preferablyreverse phase bonded silica gel with C8 or C18 alkanes, especially C18.C18 bonded reverse phase silica gel is preferred. The adsorbent used inthe process of the present invention is preferably non-polar.

The shape of the adsorbent stationary phase material may be, forexample, spherical or nonspherical beads, preferably substantiallyspherical beads. Such beads typically have a diameter of 40 to 500microns, preferably 100 to 500 microns, more preferably 250 to 500microns, even more preferably 250 to 400 microns, most preferably 250 to350 microns. These preferred particle sizes are somewhat larger thanparticle sizes of beads used in the past in simulated and actual movingbed processes. Use of larger particles enables a lower pressure ofeluent to be used in the system. This, in turn, has advantages in termsof cost savings, efficiency and lifetime of the apparatus. It hassurprisingly been found that adsorbent beads of large particle size maybe used in the process of the present invention (with their associatedadvantages) without any loss in resolution.

The adsorbent typically has a pore size of from 10 to 50 nm, preferably15 to 45 nm, more preferably 20 to 40 nm, most preferably 25 to 35 nm.

The eluent used in the process of the present invention is an aqueousalcohol. The aqueous alcohol typically comprises water and one or moreshort chain alcohols. The short chain alcohol typically has from 1 to 6carbon atoms. Examples of suitable alcohols include methanol, ethanol,n-propanol, i-propanol, n-butanol, i-butanol, s-butanol and t-butanol.Methanol and ethanol are preferred. Methanol is more preferred.

Typically, the eluent is not in a supercritical state. Typically, theeluent is a liquid.

Typically, the average water:alcohol ratio of the eluent in the entireapparatus is from 0.1:99.9 to 9:91 parts by volume, preferably from0.25:99.75 to 7:93 parts by volume, more preferably from 0.5:99.5 to6:94 parts by volume.

The eluting power of the eluent in each of the zones is typicallydifferent. Preferably, the eluting power of the eluent in the first zoneis greater than that of the eluent in the second and subsequent zones.In practice this is achieved by varying the relative amounts of waterand alcohol in each zone. Alcohols are generally more powerful desorbersthan water. Thus, the amount of alcohol in the eluent in the first zoneis typically greater than the amount of alcohol in the eluent of thesecond and subsequent zones.

In embodiments where the aqueous alcohol present in each zone has adifferent water alcohol content, the water:alcohol ratio of the eluentin the first zone is typically from 0:100 to 5:95 parts by volume,preferably from 0.1:99.9 to 2.5:97.5 parts by volume, more preferablyfrom 0.25:99.75 to 2:98 parts by volume, and most preferably from0.5:99.5 to 1.5:98.5 parts by volume. In these embodiments, thewater:alcohol ratio of the eluent in the second zone is typically from3:97 to 7:93 parts by volume, preferably from 4:96 to 6:94 parts byvolume, more preferably from 4.5:95.5 to 5.5:94.5 parts by volume.

In a particularly preferred embodiment where the aqueous alcohol presentin each zone has a different water alcohol content, the water:alcoholratio of the eluent in the first zone is from 0.5:99.5 to 1.5:98.5 partsby volume, and the water:alcohol ratio of the eluent in the second zoneis from 4.5:95:5 to 5.5:94.5 parts by volume.

In embodiments where the rate at which liquid collected via the extractand raffinate streams in each zone is recycled back into the same zoneis adjusted such that the PUFA product can be separated from differentcomponents of the feed mixture in each zone, the water:alcohol ratio ofthe eluents in each zone may be the same or different. Typically, thewater:alcohol ratio of the eluent in each zone is from 0.5:99.5 to5.5:94.5 parts by volume. In one embodiment, the water:alcohol ratio ofthe eluent in the first zone is lower than the water:alcohol ratio ofthe eluent in the second zone. In another embodiment, the water:alcoholratio of the eluent in the first zone is higher than the water:alcoholratio of the eluent in the second zone. In a further embodiment, thewater:alcohol ratio of the eluent in the first zone is the same as thewater:alcohol ratio of the eluent in the second zone.

It will be appreciated that the ratios of water and alcohol in each zonereferred to above are average ratios within the totality of the zone.

Typically, the water:alcohol ratio of the eluent in each zone iscontrolled by introducing water and/or alcohol into one or more columnsin the zones. Thus, for example, to achieve a lower water:alcohol ratioin the first zone than in the second zone, water is typically introducedmore slowly into the first zone than the second zone. In someembodiments, essentially pure alcohol and essentially pure water may beintroduced at different points in each zone. The relative flow rates ofthese two streams will determine the overall solvent profile across thezone. In other embodiments, different alcohol/water mixtures may beintroduced at different points in each zone. That will involveintroducing two or more different alcohol/water mixtures into the zone,each alcohol/water mixture having a different alcohol:water ratio. Therelative flow rates and relative concentrations of the alcohol/watermixtures in this embodiment will determine the overall solvent profileacross the zone. In other embodiments where the water:alcohol ratio ofthe eluent in each zone is the same, the same alcohol/water mixture isintroduced to each zone.

Typically, the process of the present invention is conducted at from 15to 55° C., preferably at from 20 to 40° C., more preferably at about 30°C. Thus, the process is typically carried out at room temperature, butmay be conducted at elevated temperatures.

The process of the present invention involves introducing a feed streaminto one zone (for example the first zone), collecting a firstintermediate stream enriched with the PUFA product and introducing thefirst intermediate stream into another zone (for example the secondzone). Thus, when the apparatus has two zones, the process involveseither (a) collecting a first intermediate stream from the first zoneand introducing it into the second zone, or (b) collecting a firstintermediate stream from the second zone and introducing it into thefirst zone. In this way, the PUFA product can be separated from bothmore and less polar components in a single process.

Either (a) a raffinate stream containing the PUFA product together withmore polar components is collected from a column in the first zone andintroduced to a nonadjacent column in the second zone, or (b) an extractstream containing the PUFA product together with less polar componentsis collected from a column in the second zone and introduced to anonadjacent column in the first zone.

In a particularly preferred embodiment, the apparatus has two zones, andthe process of the present invention comprises:

-   -   (i) introducing the feed mixture into the first zone, and        removing a first raffinate stream enriched with the PUFA product        and a first extract stream depleted of the PUFA product, and    -   (ii) introducing the first raffinate stream into the second        zone, removing a second raffinate stream depleted of the PUFA        product, and collecting a second extract stream to obtain the        PUFA product.

This particularly preferred embodiment is suitable for purifying EPAfrom a feed mixture.

This particularly preferred embodiment is illustrated in FIG. 2. A feedmixture F comprising the PUFA product (B) and more polar (C) and lesspolar (A) components is introduced into the first zone. In the firstzone, the less polar components (A) are removed as extract stream E1.The PUFA product (B) and more polar components (C) are removed asraffinate stream R1. Raffinate stream R1 is then introduced into thesecond zone. In the second zone, the more polar components (C) areremoved as raffinate stream R2. The PUFA product (B) is collected asextract stream E2.

This embodiment is illustrated in more detail in FIG. 4. FIG. 4 isidentical to FIG. 2, except that the points of introduction of thealcohol desorbent (D) and water (W) into each zone are shown. Thealcohol desorbent (D) and water (W) together make up the eluent. The (D)phase is typically essentially pure alcohol, but may, in certainembodiments be an alcohol/water mixture comprising mainly alcohol. The(W) phase is typically essentially pure water, but may, in certainembodiments be an alcohol/water mixture comprising mainly water, forexample a 98% water/2% methanol mixture.

A further illustration of this particularly preferred embodiment isshown in FIG. 6. Here there is no separate water injection point, andinstead an aqueous alcohol desorbent is injected at (D).

The separation into raffinate and extract stream can be aided by varyingthe desorbing power of the eluent within each zone. This can be achievedby introducing the alcohol (or alcohol rich) component of the eluent andthe water (or water rich) component at different points in each zone.Thus, typically, the alcohol is introduced upstream of the extracttake-off point and the water is introduced between the extract take-offpoint and the point of introduction of the feed into the zone, relativeto the flow of eluent in the system. This is shown in FIG. 4.

Alternatively, the separation can be aided by varying the rates at whichliquid collected via the extract and raffinate streams from the twozones is recycled back into the same zone.

Typically, in this particularly preferred embodiment, the rate at whichliquid collected via the extract stream from the first zone is recycledback into the first zone is faster than the rate at which liquidcollected via the extract stream from the second zone is recycled backinto the second zone; or the water:alcohol ratio of the eluent in thefirst zone is lower than that in the second zone.

In this particularly preferred embodiment the first raffinate stream inthe first zone is typically removed downstream of the point ofintroduction of the feed mixture into the first zone, with respect tothe flow of eluent in the first zone.

In this particularly preferred embodiment, the first extract stream inthe first zone is typically removed upstream of the point ofintroduction of the feed mixture into the first zone, with respect tothe flow of eluent in the first zone.

In this particularly preferred embodiment, the second raffinate streamin the second zone is typically removed downstream of the point ofintroduction of the first raffinate stream into the second zone, withrespect to the flow of eluent in the second zone.

In this particularly preferred embodiment, the second extract stream inthe second zone is typically collected upstream of the point ofintroduction of the first raffinate stream into the second zone, withrespect to the flow of eluent in the second zone.

Typically in this particularly preferred embodiment, the alcohol oraqueous alcohol is introduced into the first zone upstream of the pointof removal of the first extract stream, with respect to the flow ofeluent in the first zone.

Typically in this particularly preferred embodiment, when water isintroduced into the first zone, the water is introduced into the firstzone upstream of the point of introduction of the feed mixture butdownstream of the point of removal of the first extract stream, withrespect to the flow of eluent in the first zone.

Typically in this particularly preferred embodiment, the alcohol oraqueous alcohol is introduced into the second zone upstream of the pointof removal of the second extract stream, with respect to the flow ofeluent in the second zone.

Typically in this particularly preferred embodiment, when water isintroduced into the second zone, the water is introduced into the secondzone upstream of the point of introduction of the first raffinate streambut downstream of the point of removal of the second extract stream,with respect to the flow of eluent in the second zone.

In another particularly preferred embodiment, the apparatus has twozones, and the process comprises:

-   -   (i) introducing the feed mixture into the second zone, and        removing a first raffinate stream depleted of the PUFA product        and a first extract stream enriched in the PUFA product, and    -   (ii) introducing the first extract stream into the first zone,        removing a second extract stream depleted of the PUFA product,        and collecting a second raffinate stream to obtain the PUFA        product.

This particularly preferred embodiment is suitable for purifying DHAfrom a feed mixture.

This embodiment is illustrated in FIG. 3. A feed mixture F comprisingthe PUFA product (B) and more polar (C) and less polar (A) components isintroduced into the second zone. In the second zone, the more polarcomponents (C) are removed as raffinate stream R1. The PUFA product (B)and less polar components (A) are collected as extract stream E1.Extract stream E1 is then introduced to the first zone. In the firstzone, the less polar components (A) are removed as extract stream E2.The PUFA product (B) is collected as raffinate stream R2.

This embodiment is illustrated in more detail in FIG. 5. FIG. 5 isidentical to FIG. 3, except that the points of introduction of the shortchain alcohol desorbent (D) and water (W) into each zone are shown. Asabove, the (D) phase is typically essentially pure alcohol, but may, incertain embodiments be an alcohol/water mixture comprising mainlyalcohol. The (W) phase is typically essentially pure water, but may, incertain embodiments be an alcohol/water mixture comprising mainly water,for example a 98% water/2% methanol mixture.

A further illustration of this particularly preferred embodiment isshown in FIG. 7. Here there is no separate water injection point, andinstead an aqueous alcohol desorbent is injected at (D).

Typically in this embodiment, the rate at which liquid collected via theraffinate stream from the second zone is reintroduced into the secondzone is faster than the rate at which liquid collected via the raffinatestream from the first zone is reintroduced into the first zone; or thewater:alcohol ratio of the eluent in the first zone is lower than thatin the second zone.

In this second particularly preferred embodiment, the first raffinatestream in the second zone is typically removed downstream of the pointof introduction of the feed mixture into the second zone, with respectto the flow of eluent in the second zone.

In this second particularly preferred embodiment, the first extractstream in the second zone is typically collected upstream of the pointof introduction of the feed mixture into the second zone, with respectto the flow of eluent in the second zone.

In this second particularly preferred embodiment, the second raffinatestream in the first zone is typically collected downstream of the pointof introduction of the first extract stream into the first zone, withrespect to the flow of eluent in the first zone.

In this second particularly preferred embodiment, the second extractstream in the first zone is typically removed upstream of the point ofintroduction of the first extract stream into the first zone, withrespect to the flow of eluent in the first zone.

Typically in this second particularly preferred embodiment, the alcoholor aqueous alcohol is introduced into the second zone upstream of thepoint of removal of the first extract stream, with respect to the flowof eluent in the second zone.

Typically in this second particularly preferred embodiment, when wateris introduced into the second zone, the water is introduced into thesecond zone upstream of the point of introduction of the feed mixturebut downstream of the point of removal of the first extract stream, withrespect to the flow of eluent in the second zone.

Typically in this second particularly preferred embodiment, the alcoholor aqueous alcohol is introduced into the first zone upstream of thepoint of removal of the second extract stream, with respect to the flowof eluent in the first zone.

Typically in this second particularly preferred embodiment, when wateris introduced into the first zone, the water is introduced into thefirst zone upstream of the point of introduction of the first raffinatestream but downstream of the point of removal of the second extractstream, with respect to the flow of eluent in the first zone.

In a preferred embodiment of the invention, the simulated or actualmoving bed chromatography apparatus consists of fifteen chromatographiccolumns. These are referred to as columns 1 to 15. The fifteen columnsare arranged in series so that the bottom of column 1 is linked to thetop of column 2, the bottom of column 2 is linked to the top of column 3etc. This may optionally be via a holding container, with a recyclestream into the next column. The flow of eluent through the system isfrom column 1 to column 2 to column 3 etc. The flow of adsorbent throughthe system is from column 15 to column 14 to column 13 etc.

In a most preferred embodiment, the first zone typically consists ofeight adjacent columns, columns 1 to 8, which are connected as discussedabove. In this most preferred embodiment, the second zone typicallyconsists of seven columns, columns 9 to 15, which are connected asdiscussed above. For the avoidance of doubt, the bottom of column 8 inthe first zone is linked to the top of column 9 in the second zone.

A most preferred embodiment is illustrated in FIG. 8. A feed mixture Fcomprising the PUFA product (B) and more polar (C) and less polar (A)components is introduced into the top of column 5 in the first zone.Alcohol desorbent is introduced into the top of column 1 in the firstzone. Water is introduced into the top of column 4 in the first zone. Inthe first zone, the less polar components (A) are removed as extractstream E1 from the bottom of column 2. The PUFA product (B) and morepolar components (C) are removed as raffinate stream R1 from the bottomof column 7. Raffinate stream R1 is then introduced into the second zoneat the top of column 13. Alcohol desorbent is introduced into the top ofcolumn 9 in the second zone. Water is introduced into the top of column12 in the second zone. In the second zone, the more polar components (C)are removed as raffinate stream R2 at the bottom of column 15. The PUFAproduct (B) is collected as extract stream E2 at the bottom of column10.

In this most preferred embodiment, alcohol is typically introduced intothe top of column 1 in the first zone.

In this most preferred embodiment, water is typically introduced intothe top of column 4 in the first zone.

In this most preferred embodiment, alcohol is typically introduced intothe top of column 9 in the second zone.

In this most preferred embodiment, alcohol is typically introduced intothe top of column 12 in the second zone.

In this most preferred embodiment, the feed stream is typicallyintroduced into the top of column 5 in the first zone.

In this most preferred embodiment, a first raffinate stream is typicallycollected from the bottom of column 7 in the first zone and introducedinto the top of column 13 in the second zone. The first raffinate streammay optionally be collected in a container before being introduced intocolumn 13.

In this most preferred embodiment, a first extract stream is typicallyremoved from the bottom of column 2 in the first zone. The first extractstream may optionally be collected in a container and reintroduced intothe top of column 3 in the first zone.

In this most preferred embodiment, a second raffinate stream istypically removed from the bottom of column 15 in the second zone.

In this most preferred embodiment, a second extract stream is typicallycollected from the bottom of column 10 in the second zone. This secondextract stream typically contains the purified PUFA product. The secondextract stream may optionally be collected in a container andreintroduced into the top of column 11 in the second zone.

Typically, in this most preferred embodiment, the water:alcohol ratio inthe first zone is lower than the water:alcohol ratio in the second zone.

A further most preferred embodiment is illustrated in FIG. 9. A feedmixture F comprising the PUFA product (B) and more polar (C) and lesspolar (A) components is introduced into the top of column 5 in the firstzone. Aqueous alcohol desorbent is introduced into the top of column 1in the first zone. In the first zone, the less polar components (A) areremoved as extract stream E1 from the bottom of column 2. The PUFAproduct (B) and more polar components (C) are removed as raffinatestream R1 from the bottom of column 7. Raffinate stream R1 is thenintroduced into the second zone at the top of column 12. Aqueous alcoholdesorbent is introduced into the top of column 9 in the second zone. Inthe second zone, the more polar components (C) are removed as raffinatestream R2 at the bottom of column 14. The PUFA product (B) is collectedas extract stream E2 at the bottom of column 10.

In this most preferred embodiment, aqueous alcohol is typicallyintroduced into the top of column 1 in the first zone.

In this most preferred embodiment, aqueous alcohol is typicallyintroduced into the top of column 9 in the second zone.

In this most preferred embodiment, the feed stream is typicallyintroduced into the top of column 5 in the first zone.

In this most preferred embodiment, a first raffinate stream is typicallycollected from the bottom of column 7 in the first zone and introducedinto the top of column 12 in the second zone. The first raffinate streammay optionally be collected in a container before being introduced intocolumn 12.

In this most preferred embodiment, a first extract stream is typicallyremoved from the bottom of column 2 in the first zone. The first extractstream may optionally be collected in a container and a portionreintroduced into the top of column 3 in the first zone. The rate ofrecycle of liquid collected via the extract stream from the first zoneback into the first zone is the rate at which liquid is pumped from thiscontainer into the top of column 3.

In this most preferred embodiment, a second raffinate stream istypically removed from the bottom of column 14 in the second zone.

In this most preferred embodiment, a second extract stream is typicallycollected from the bottom of column 10 in the second zone. This secondextract stream typically contains the purified PUFA product. The secondextract stream may optionally be collected in a container and a portionreintroduced into the top of column 11 in the second zone. The rate ofrecycle of liquid collected via the extract stream from the second zoneback into the second zone is the rate at which liquid is pumped fromthis container into the top of column 11.

In this most preferred embodiment, the rate at which liquid collectedvia the extract stream from the first zone is recycled back into thefirst zone is typically faster than the rate at which liquid collectedvia the extract stream from the second zone is recycled back into thesecond zone.

In this most preferred embodiment, the aqueous alcohol eluent issubstantially the same in each zone.

In a further preferred embodiment of the invention, the simulated oractual moving bed chromatography apparatus consists of nineteenchromatographic columns. These are referred to as columns 1 to 19. Thefifteen columns are arranged in series so that the bottom of column 1 islinked to the top of column 2, the bottom of column 2 is linked to thetop of column 3 etc. The flow of eluent through the system is fromcolumn 1 to column 2 to column 3 etc. The flow of adsorbent through thesystem is from column 19 to column 18 to column 17 etc.

In this embodiment, the first zone typically consists of ten adjacentcolumns, columns 1 to 10, which are connected as discussed above. Thesecond zone typically consists of eight columns, columns 11 to 19, whichare connected as discussed above.

This further preferred embodiment is illustrated in FIG. 10. A feedmixture F comprising the PUFA product (B) and more polar (C) and lesspolar (A and A′) components is introduced into the top of column 7 inthe first zone. A first desorbent (D1) comprising 100% alcohol isintroduced into the top of column 1 in the first zone. A seconddesorbent (D2) comprising a water/alcohol mixture (preferably 2%methanol and 98% water) is introduced into the top of column 5 in thefirst zone. In the first zone, less polar components (A′) and (A) areremoved as extract streams E1′ and E1 from the bottoms of columns 1 and4, respectively. The PUFA product (B) and more polar components (C) areremoved as raffinate stream R1 from the bottom of column 10. Raffinatestream R1 is then introduced into the second zone at the top of column17. A second desorbent (D2) comprising a water/alcohol mixture(preferably 2% methanol and 98% water) is introduced into the top ofcolumn 11 in the second zone. In the second zone, the more polarcomponents (C) are removed as raffinate stream R2 at the bottom ofcolumn 19. The PUFA product (B) is collected as extract stream E2 at thebottom of column 14.

In this preferred embodiment, alcohol is typically introduced into thetop of column 1 in the first zone.

In this preferred embodiment, a 2% MeOH/98% water mixture is typicallyintroduced into the top of column 5 in the first zone.

In this preferred embodiment, a 2% MeOH/98% water mixture is typicallyintroduced into the top of column 11 in the second zone.

In this preferred embodiment, the feed stream is typically introducedinto the top of column 7 in the first zone.

In this preferred embodiment, a first raffinate stream is typicallycollected from the bottom of column 10 in the first zone and introducedinto the top of column 17 in the second zone. The first raffinate streammay optionally be collected in a container before being introduced intocolumn 17.

In this preferred embodiment, extract streams are typically removed fromthe bottoms of columns 1 and 4 in the first zone. The extract streamcollected from the bottom or column 4 may optionally be collected in acontainer and reintroduced into the top of column 5 in the first zone.

In this preferred embodiment, a second raffinate stream is typicallyremoved from the bottom of column 19 in the second zone.

In this preferred embodiment, a second extract stream is typicallycollected from the bottom of column 14 in the second zone. This secondextract stream typically contains the purified PUFA product. The secondextract stream may optionally be collected in a container andreintroduced into the top of column 15 in the second zone.

Typically, in this most preferred embodiment, the water:alcohol ratio inthe first zone is lower than the water:alcohol ratio in the second zone.

The process of the invention allows much higher purities of PUFA productto be achieved than have been possible with conventional chromatographictechniques. PUFA products produced by the process of the invention alsohave particularly advantageous impurity profiles, which are quitedifferent from those observed in oils prepared by known techniques. Thepresent invention therefore also relates to compositions comprising aPUFA product, for example one obtainable by the process of the presentinvention.

Thus, in one embodiment the present invention also provides acomposition comprising a PUFA product, wherein the PUFA product is EPA,the PUFA product is present in an amount greater than 93 wt %, and thetotal content of ω-6 polyunsaturated fatty acids is up to 0.40 wt %.

As used herein, the wt % of a component is relative to the total weightof the composition.

The PUFA product and ω-6 PUFAs are optionally in the form of their alkylesters, typically ethyl esters. Preferably, the EPA PUFA product is inthe form of its ethyl ester.

Typically, in this embodiment the EPA PUFA product is present in anamount greater than 94 wt %, preferably greater than 95 wt %, morepreferably greater than 96 wt %, even more preferably greater than 97 wt%, and most preferably greater than 98 wt %.

The total content of ω-6 polyunsaturated fatty acids in this embodimentis up to 0.40 wt %. Thus, typically, the composition comprises an amountof ω-6 polyunsaturated fatty acids in up to this amount. Typically, thetotal content of ω-6 polyunsaturated fatty acids is up to 0.35 wt %,preferably up to 0.3 wt %, more preferably up to 0.25 wt %, and mostpreferably up to 0.22 wt %. Typically, the total content of ω-6polyunsaturated fatty acids is 0.05 wt % or greater, preferably 0.1 wt %or greater.

Typically, in this embodiment the content of arachidonic acid is up to0.25 wt %, preferably up to 0.24 wt %, more preferably up to 0.23 wt %,and most preferably up to 0.22 wt %. Thus, typically, the compositioncomprises an amount of arachidonic acid in up to these amounts.Typically, the total content of arachidonic acid is 0.05 wt % orgreater, preferably 0.1 wt % or greater.

Typically, in this embodiment the total content of ω-3 polyunsaturatedfatty acids is greater than 97 wt %, preferably greater than 97.5 wt %,more preferably greater than 97.9 wt %. In certain embodiments, thetotal content of ω-3 polyunsaturated fatty acids is greater than 99 wt%.

Typically, in this embodiment the total content of DHA is up to 1 wt %,preferably up to 0.6 wt %, more preferably up to 0.3 wt %, mostpreferably up to 0.2 wt %. Thus, typically, the composition comprises anamount of DHA in up to these amounts. Typically, the total content ofDHA is 0.05 wt % or greater, preferably 0.1 wt % or greater.

Typically, in this embodiment the total content of DHA is up to 0.2 wt%, preferably up to 0.175 wt %, more preferably up to 0.16 wt %. Thus,typically, the composition comprises an amount of DHA in up to theseamounts. Typically, the total content of DHA is 0.05 wt % or greater,preferably 0.1 wt % or greater.

Typically, in this embodiment the total content of α-linolenic acid isup to 1 wt %, preferably up to 0.6 wt %, more preferably up to 0.3 wt %.Thus, typically, the composition comprises an amount of α-linolenic acidin up to these amounts. Typically, the total content of α-linolenic acidis 0.05 wt % or greater, preferably 0.1 wt % or greater.

Typically, in this embodiment the total content of α-linolenic acid isup to 0.35 wt %, preferably up to 0.3 wt %, more preferably up to 0.29wt %. Thus, typically, the composition comprises an amount ofα-linolenic acid in up to these amounts. Typically, the total content ofα-linolenic acid is 0.05 wt % or greater, preferably 0.1 wt % orgreater.

Typically, in this embodiment the total content of stearidonic acid isup to 1 wt %, preferably up to 0.6 wt %, more preferably up to 0.3 wt %.Thus, typically, the composition comprises an amount of stearidonic acidin up to these amounts. Typically, the total content of stearidonic acidis 0.05 wt % or greater, preferably 0.1 wt % or greater.

Typically, in this embodiment the total content of stearidonic acid isup to 0.4 wt %, preferably up to 0.35 wt %, more preferably up to 0.34wt %. Thus, typically, the composition comprises an amount ofstearidonic acid in up to these amounts.

Typically, the total content of stearidonic acid is 0.05 wt % orgreater, preferably 0.1 wt % or greater.

Typically, in this embodiment the total content of eicosatetraenoic acidis up to 1 wt %, preferably up to 0.75 wt %, more preferably up to 0.5wt %. Thus, typically, the composition comprises an amount ofeicosatetraenoic acid in up to these amounts. Typically, the totalcontent of eicosatetraenoic acid is 0.05 wt % or greater, preferably 0.1wt % or greater.

Typically, in this embodiment the total content of eicosatetraenoic acidis up to 0.5 wt %, preferably up to 0.475 wt %, more preferably up to0.46 wt %. Thus, typically, the composition comprises an amount ofeicosatetraenoic acid in up to these amounts. Typically, the totalcontent of eicosatetraenoic acid is 0.05 wt % or greater, preferably 0.1wt % or greater.

Typically, in this embodiment the total content of docosapentaenoic acidis up to 1 wt %, preferably up to 0.6 wt %, more preferably up to 0.3 wt%. Thus, typically, the composition comprises an amount ofdocosapentaenoic acid in up to these amounts. Typically, the totalcontent of docosapentaenoic acid is 0.05 wt % or greater, preferably 0.1wt % or greater.

Typically, in this embodiment the total content of docosapentaenoic acidis up to 0.4 wt %, preferably up to 0.35 wt %, more preferably up to0.33 wt %. Thus, typically, the composition comprises an amount ofdocosapentaenoic acid in up to these amounts. Typically, the totalcontent of docosapentaenoic acid is 0.05 wt % or greater, preferably 0.1wt % or greater.

In this embodiment, the composition preferably comprises greater than96.5 wt % EPA, up to 1 wt % DHA, up to 1 wt % α-linolenic acid, up to 1wt % stearidonic acid, up to 1 wt % eicosatetraenoic acid, up to 1 wt %docosapentaenoic acid, and up to 0.25 wt % arachidonic acid.

In this embodiment, the composition preferably comprises greater than96.5 wt % EPA, up to 0.2 wt % DHA, up to 0.3 wt % α-linolenic acid, upto 0.4 wt % stearidonic acid, up to 0.5 wt % eicosatetraenoic acid, upto 0.35 wt % docosapentaenoic acid, and up to 0.25 wt % arachidonicacid.

In this embodiment, the composition more preferably comprises from 96.5to 99 wt % EPA, up to 0.6 wt % DHA, up to 0.6 wt % α-linolenic acid,from 0.15 to 0.6 wt % stearidonic acid, from 0.1 to 0.75 wt %eicosatetraenoic acid, up to 0.6 wt % docosapentaenoic acid, and up to0.6 wt % arachidonic acid.

In this embodiment, the composition more preferably comprises from 96.5to 99 wt % EPA, up to 0.2 wt % DHA, up to 0.3 wt % α-linolenic acid,from 0.15 to 0.4 wt % stearidonic acid, from 0.1 to 0.5 wt %eicosatetraenoic acid, up to 0.35 wt % docosapentaenoic acid, and up to0.25 wt % arachidonic acid.

In this embodiment, the composition most preferably comprises from 98 to99 wt % EPA, from 0.1 to 0.3 wt % DHA, from 0.3 to 0.35 wt % stearidonicacid, from 0.1 to 0.3 wt % eicosatetraenoic acid, and from 0.3 to 0.35wt % docosapentaenoic acid.

In this embodiment, the composition most preferably comprises from 96.5to 99 wt % EPA, from 0.1 to 0.5 wt % DHA, from 0.1 to 0.5 wt %stearidonic acid, from 0.1 to 0.5 wt % eicosatetraenoic acid, from 0.1to 0.5 wt % docosapentaenoic acid, and from 0.1 to 0.3 wt % arachidonicacid.

In this embodiment, the composition most preferably comprises from 98 to99 wt % EPA, from 0.1 to 0.2 wt % DHA, from 0.3 to 0.35 wt % stearidonicacid, from 0.1 to 0.2 wt % eicosatetraenoic acid, and from 0.3 to 0.35wt % docosapentaenoic acid.

In this embodiment, the composition most preferably comprises from 96.5to 97.5 wt % EPA, from 0.25 to 0.35 wt % α-linolenic acid, from 0.18 to0.24 wt % stearidonic acid, from 0.4 to 0.46 wt % eicosatetraenoic acid,and from 0.15 to 0.25 wt % arachidonic acid.

Typically, in this embodiment, the content of isomeric impurities is upto 1.5 wt %. Typically, the content of isomeric impurities is up to 1 wt%, preferably up to 0.5 wt %, more preferably up to 0.25 wt %, even morepreferably up to 0.25 wt %, and most preferably up to 0.1 wt %.

In a further embodiment, the present invention also provides acomposition comprising a PUFA product, wherein the PUFA product is amixture of EPA and DHA, wherein (i) the total content of EPA and DHA is80 wt % or greater, (ii) the content of EPA is from 41 to 60 wt % andthe content of DHA is from 16 to 48 wt %, and (iii) the total content ofω-3 polyunsaturated fatty acids is 94 wt % or greater and/or the totalcontent of ω-6 polyunsaturated fatty acids is up to 4 wt %.

The PUFA product, ω-3 and ω-6 PUFAs are optionally in the form of theiralkyl esters, typically ethyl esters. Preferably, the EPA/DHA PUFAproduct is in the form of its ethyl esters.

Thus, in this further embodiment the composition is typically acomposition comprising a PUFA product, wherein the PUFA product is amixture of EPA and DHA, wherein (i) the total content of EPA and DHA is80 wt % or greater, (ii) the content of EPA is from 41 to 60 wt % andthe content of DHA is from 16 to 48 wt %, and (iii) the total content ofω-3 polyunsaturated fatty acids is 94 wt % or greater.

Alternatively, in this further embodiment the composition is acomposition comprising a PUFA product, wherein the PUFA product is amixture of EPA and DHA, wherein (i) the total content of EPA and DHA is80 wt % or greater, (ii) the content of EPA is from 41 to 60 wt % andthe content of DHA is from 16 to 48 wt %, and (iii) the total content ofω-6 polyunsaturated fatty acids is up to 4 wt %.

Typically, in this further embodiment the total content of EPA and DHAis 82 wt % or greater, preferably 83 wt % or greater, more preferably 84wt % or greater, even more preferably 85 wt % or greater, and mostpreferably 86 wt % or greater.

Typically, in this further embodiment the content of EPA is from 41 to60 wt %, preferably from 45 to 60 wt %, more preferably from 47 to 60 wt%, even more preferably from 47 to 57 wt %, and most preferably from 50to 55 wt %.

Typically, in this further embodiment the content of DHA is from 16 to48 wt %, preferably from 20 to 45 wt %, more preferably from 25 to 42 wt%, even more preferably from 28 to 38 wt %, and most preferably from 30to 35 wt %.

Typically, in this further embodiment the total content of ω-3polyunsaturated fatty acids is 94 wt % or greater, preferably 95 wt % orgreater, more preferably 96 wt % or greater, and most preferably 97 wt %or greater.

Typically, in this further embodiment the total content of α-linolenicacid is up to 0.4 wt %, preferably up to 0.35 wt %, more preferably upto 0.31 wt %. Thus, typically, the composition comprises an amount ofα-linolenic acid in up to these amounts. Typically, the total content ofα-linolenic acid is 0.05 wt % or greater, preferably 0.1 wt % orgreater, more preferably 0.2 wt % or greater, and even preferably from0.2 to 0.4 wt %.

Typically, in this further embodiment the total content of stearidonicacid is up to 1.9 wt %, preferably up to 1.5 wt %, more preferably up to1.25 wt %. Thus, typically, the composition comprises an amount ofstearidonic acid in up to these amounts. Typically, the total content ofstearidonic acid is 0.05 wt % or greater, preferably 0.1 wt % orgreater.

Typically, in this further embodiment the total content ofeicosatetraenoic acid is up to 2.0 wt %, preferably up to 1.9 wt %.Thus, typically, the composition comprises an amount of eicosatetraenoicacid in up to these amounts. Typically, the total content ofeicosatetraenoic acid is 0.05 wt % or greater, preferably 0.1 wt % orgreater, more preferably 1.0 wt % or greater, and even more preferablyfrom 1.0 to 1.9 wt %.

Typically, in this further embodiment the total content ofeicosapentaenoic acid is up to 3.0 wt %, preferably up to 2.75 wt %.Thus, typically, the composition comprises an amount of eicosapentaenoicacid in up to these amounts. Typically, the total content ofeicosapentaenoic acid is 0.05 wt % or greater, preferably 0.1 wt % orgreater, more preferably 2 wt % or greater, and even more preferablyfrom 2 to 2.75 wt %.

Typically, in this further embodiment the total content ofdocosapentaenoic acid is up to 6 wt %, preferably up to 5.5 wt %, morepreferably up to 5.25 wt %. Thus, typically, the composition comprisesan amount of docosapentaenoic acid in up to these amounts. Typically,the total content of docosapentaenoic acid is 0.05 wt % or greater,preferably 0.1 wt % or greater, more preferably 4 wt % or greater, andeven more preferably from 4 to 5.25 wt %.

The total content of ω-6 polyunsaturated fatty acids in this furtherembodiment is typically up to 4 wt %. Thus, typically, the compositioncomprises an amount of ω-6 polyunsaturated fatty acids up to theseamounts. Typically, the total content of ω-6 polyunsaturated fatty acidsis up to 3.75 wt %, preferably up to 3.5 wt %, more preferably up to3.25 wt %, even more preferably up to 3 wt %, most preferably up to 2.85wt %. Typically, the total content of ω-6 polyunsaturated fatty acids is0.05 wt % or greater, preferably 0.1 wt % or greater.

Typically, in this further embodiment the total content of linoleic acidis up to 0.5 wt %, preferably up to 0.4 wt %, more preferably up to 0.25wt %. Thus, typically, the composition comprises an amount of linoleicacid in up to these amounts. Typically, the total content of linoleicacid is 0.05 wt % or greater, preferably 0.1 wt % or greater, morepreferably 0.15 wt % or greater, and even more preferably from 0.15 to0.25 wt %.

Typically, in this further embodiment the total content ofgamma-linolenic acid is up to 0.19 wt %, preferably up to 0.15 wt %,more preferably up to 0.1 wt %. Thus, typically, the compositioncomprises an amount of gamma-linolenic acid in up to these amounts.Typically, the total content of gamma-linolenic acid is 0.05 wt % orgreater, preferably 0.1 wt % or greater.

Typically, in this further embodiment the total content ofdihommo-gamma-linolenic acid is up to 0.1 wt %. Thus, typically, thecomposition comprises an amount of dihommo-gamma-linolenic acid in up tothese amounts. Typically, the total content of dihommo-gamma-linolenicacid is 0.05 wt % or greater.

Typically, in this further embodiment the total content of arachidonicacid is up to 2.5 wt %, preferably up to 2.25 wt %, more preferably upto 2.1 wt %. Thus, typically, the composition comprises an amount ofarachidonic acid in up to these amounts. Typically, the total content ofarachidonic acid is 0.05 wt % or greater, preferably 0.1 wt % orgreater.

Typically, in this further embodiment the total content of adrenic acidis up to 0.1 wt %. Thus, typically, the composition comprises an amountof adrenic acid in up to this amount. Typically, the total content ofadrenic acid is 0.05 wt % or greater.

Typically, in this further embodiment the total content ofdocosapentaenoic (ω-6) acid is up to 0.9 wt %, preferably up to 0.75 wt%. More preferably up to 0.65 wt %. Thus, typically, the compositioncomprises an amount of docosapentaenoic (ω-6) acid in up to theseamounts. Typically, the total content of docosapentaenoic (ω-6) acid is0.05 wt % or greater, preferably 0.1 wt % or greater.

In this further embodiment, the composition preferably comprises from 50to 55 wt % EPA, from 30 to 35 wt % DHA, up to 0.4 wt % α-linolenic acid,up to 1.25 wt % stearidonic acid, up to 1.9 wt % eicosatetraenoic acid,up to 2.75 wt % eicosapentaenoic acid, up to 5.25 wt % docosapentaenoicacid, up to 0.25 wt % linoleic acid, up to 0.1 wt % gamma-linolenicacid, up to 0.1 wt % dihommo-gamma-linolenic acid, up to 2.1 wt %arachidonic acid, up to 0.1 wt % adrenic acid, and up to 0.75 wt %docosapentaenoic (ω-6) acid.

In this further embodiment, the composition more preferably comprisesfrom 50 to 55 wt % EPA, from 30 to 35 wt % DHA, from 0.2 to 0.4 wt %α-linolenic acid, up to 1.25 wt % stearidonic acid, from 1.0 to 1.9 wt %eicosatetraenoic acid, from 2 to 2.75 wt % eicosapentaenoic acid, from 4to 5.25 wt % docosapentaenoic acid, from 0.15 to 0.25 wt % linoleicacid, up to 0.1 wt % gamma-linolenic acid, up to 0.1 wt %dihommo-gamma-linolenic acid, up to 2.1 wt % arachidonic acid, up to 0.1wt % adrenic acid, and up to 0.75 wt % docosapentaenoic (ω-6) acid.

Typically, in this further embodiment, the content of isomericimpurities is up to 1.5 wt %. Typically, the content of isomericimpurities is up to 1 wt %, preferably up to 0.5 wt %, more preferablyup to 0.25 wt %, even more preferably up to 0.25 wt %, and mostpreferably up to 0.1 wt %.

The inventors have also surprisingly found that oils can be producedwith a reduced amount of environmental pollutants, compared with knownoils. Thus, in a still further embodiment the present invention alsoprovides a composition comprising a PUFA product, as defined herein,wherein (a) the total amount of polyaromatic hydrocarbons in thecomposition is up to 0.89 μg/kg, (b) the total amount of dioxins,furans, dibenzeno-para-dioxins and polychlorinated dibenzofurans is upto 0.35 pg/g (c) the total amount of polychlorinated biphenyls is up to0.0035 mg/kg, and/or (d) the total amount of dioxins, furans,dibenzeno-para-dioxins, polychlorinated dibenzofurans and dioxin-likepolychlorinated biphenyls is up to 1 pg/g.

Typically, the present invention provides a composition comprising aPUFA product, as defined herein, wherein (a) the total amount ofpolyaromatic hydrocarbons in the composition is up to 0.89 μg/kg, (b)the total amount of dioxins, furans, dibenzeno-para-dioxins andpolychlorinated dibenzofurans is up to 0.35 pg/g, and/or (d) the totalamount of dioxins, furans and dioxin-like polychlorinated biphenyls isup to 1 pg/g.

The total amount of polyaromatic hydrocarbons in this still furtherembodiment in the composition is up to 0.89 μg/kg. Thus, typically, thecomposition comprises an amount of polyaromatic hydrocarbons up to thisamount. Typically, the total amount of polyaromatic hydrocarbons in thecomposition is up to 0.85 μg/kg, preferably up to 0.8 μg/kg, morepreferably up to 0.7 μg/kg, even more preferably up to 0.6 μg/kg, stillmore preferably up to 0.5 μg/kg, yet more preferably up to 0.4 μg/kg,yet more preferably up to 0.3 μg/kg, yet more preferably up to 0.2μg/kg, yet more preferably up to 0.1 μg/kg, and most preferably up to0.05 μg/kg.

Typical polyaromatic hydrocarbons are well known to one skilled in theart and include acenaphthene, acenaphthylene, anthracene,benz[a]anthracene, benzo[a]pyrene, benzo[e]pyrene, benzo[b]fluoranthene,benzo[ghi]perylene, benzo[j]fluoranthene, benzo[k]fluoranthene,chrysene, dibenz(ah)anthracene, fluoranthene, fluorine,indeno(1,2,3-cd)pyrene, phenanthrene, pyrene, coronene, corannulene,tetracene, naphthalene, pentacene, triphenylene, and ovalene. Typically,the amounts referred to above refer to the content of benzo[a]pyrene.

The total amount of dioxins, furans, dibenzeno-para-dioxins andpolychlorinated dibenzofurans in this still further embodiment is up to0.35 pg/g. Thus, typically, the composition comprises an amount ofdioxins, furans, dibenzeno-para-dioxins and polychlorinateddibenzofurans up to this amount. Typically, the total amount of dioxins,furans, dibenzeno-para-dioxins and polychlorinated dibenzofurans is upto 0.325 pg/g, preferably up to 0.3 pg/g, more preferably up to 0.275pg/g, even more preferably up to 0.25 pg/g, still more preferably up to0.225 pg/g, yet more preferably up to 0.2 pg/g, and most preferably upto 0.185 pg/g. These amounts are expressed in World Health Organisation(WHO) toxic equivalents using WHO-toxic equivalent factors (TEFs).WHO-toxic equivalent factors are well known to the person skilled in theart.

Dioxins, furans, dibenzeno-para-dioxins (PCDDs) and polychlorinateddibenzofurans (PCDFs) are well known to the skilled person. Typically,these are as defined in Community regulations (EC) No. 1881/2006 and1883/2006 the entirety of which is incorporated herein by reference.

The PCDDs, PCDFs and Dioxin-like PCBs defined in Community regulations(EC) No. 1881/2006 and 1883/2006 together with their TEF values are asfollows.

Congener TEF value Dibenzo-p-dioxins (PCDDs) 2,3,7,8-TCDD 11,2,3,7,8-PeCDD 1 1,2,3,4,7,8-HxCDD 0.1 1,2,3,6,7,8-HxCDD 0.11,2,3,7,8,9-HxCDD 0.1 1,2,3,4,6,7,8-HpCDD 0.01 OCDD 0.0001 Dibenzofurans(PCDFs) 2,3,7,8-TCDF 0.1 1,2,3,7,8-PeCDF 0.05 2,3,4,7,8-PeCDF 0.51,2,3,4,7,8-HxCDF 0.1 1,2,3,6,7,8-HxCDF 0.1 1,2,3,7,8,9-HxCDF 0.12,3,4,6,7,8-HxCDF 0.1 1,2,3,4,6,7,8-HpCDF 0.01 1,2,3,4,7,8,9-HpCDF 0.01OCDF 0.0001 Dioxin-like PCBs: Non-ortho PCBs + Mono-ortho PCBs Non-orthoPCBs PCB 77 0.0001 PCB 81 0.0001 PCB 126 0.1 PCB 169 0.01 Mono-orthoPCBs PCB 105 0.0001 PCB 114 0.0005 PCB 118 0.0001 PCB 123 0.0001 PCB 1560.0005 PCB 157 0.0005 PCB 167 0.00001 PCB 189 0.0001 Abbreviations used:‘T’ = tetra; ‘Pe’ = penta: ‘Hx’ = hexa; ‘Hp’ = hepta; ‘O’ = octa; ‘CDD’= chlorodibenzodioxin; ‘CDF’ = chlorodibenzofuran; ‘CB’ =chlorobiphenyl.

Typically, the quantity of PCDDs, PCDFs and Dioxin-like PCBs isdetermined according to the method set out in Community regulations (EC)No. 1881/2006 and 1883/2006.

The total amount of polychlorinated biphenyls in this still furtherembodiment is up to 0.0035 mg/kg. Thus, typically, the compositioncomprises an amount of polychlorinated biphenyls up to this amount.Typically, the total amount of polychlorinated biphenyls is up to 0.003mg/kg, preferably up to 0.0025 mg/kg, more preferably up to 0.002 mg/kg,even more preferably up to 0.0015 mg/kg, still more preferably up to0.001 mg/kg, yet more preferably up to 0.00075 mg/kg, and mostpreferably up to 0.0007 mg/kg.

Polychlorobiphenyls (PCBs) are well known in the art and includesbiphenyl, monochlorobiphenyl, dichlorobiphenyl, trichlorobiphenyl,tetrachlorobiphenyl, pentachlorobiphenyl, hexachlorobiphenyl,heptachlorobiphenyl, octachlorobiphenyl, nonachlorobiphenyl, anddecachlorobiphenyl.

The total amount of dioxins, furans, dibenzeno-para-dioxins,polychlorinated dibenzofurans and dioxin-like polychlorinated biphenylsin this still further embodiment is up to 1 pg/g, Thus, typically, thecomposition comprises an amount of dioxins, furans,dibenzeno-para-dioxins, polychlorinated dibenzofurans and dioxin-likepolychlorinated biphenyls in up to this amount. Typically, the totalamount of dioxins, furans, dibenzeno-para-dioxins, polychlorinateddibenzofurans and dioxin-like polychlorinated biphenyls is up to 0.75pg/g, preferably up to 0.5 pg/g, more preferably up to 0.45 pg/g, evenmore preferably up to 0.4 pg/g, still more preferably up to 0.35 pg/g,and most preferably up to 0.3 pg/g.

Dioxins, furans, dibenzeno-para-dioxins (PCDDs), polychlorinateddibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls are wellknown to the skilled person. Typically, these are as defined inCommunity regulation (EC) No. 1881/2006 and 1883/2006 the entirety ofwhich is incorporated herein by reference.

The PCDDs, PCDFs and Dioxin-like PCBs defined in Community regulation(EC) No. 1881/2006 and 1883/2006 together with their TEF values are asdefined above.

In this still further embodiment, preferably (a) the total amount ofpolyaromatic hydrocarbons in the composition is up to 0.05 mg/kg, (b),the total amount of dioxins, furans, dibenzeno-para-dioxins andpolychlorinated dibenzofurans is up to 0.2 pg/g (c) the total amount ofpolychlorinated biphenyls is up to 0.0015 mg/kg, and/or (d) the totalamount of dioxins, furans, dibenzeno-para-dioxins and polychlorinateddibenzofurans and dioxin-like polychlorinated biphenyls is up to 0.3pg/g.

In this still further embodiment, preferably (a) the total amount ofpolyaromatic hydrocarbons in the composition is up to 0.05 mg/kg, (b),the total amount of dioxins, furans, dibenzeno-para-dioxins andpolychlorinated dibenzofurans is up to 0.2 pg/g and/or (d) the totalamount of dioxins, furans, dibenzeno-para-dioxins and polychlorinateddibenzofurans and dioxin-like polychlorinated biphenyls is up to 0.3pg/g.

In this still further embodiment, more preferably (a) the total amountof polyaromatic hydrocarbons in the composition is up to 0.05 μg/kg,(b), the total amount of dioxins, furans, dibenzeno-para-dioxins andpolychlorinated dibenzofurans is up to 0.2 pg/g (c) the total amount ofpolychlorinated biphenyls is up to 0.0015 mg/kg, and (d) the totalamount of dioxins, furans, dibenzeno-para-dioxins and polychlorinateddibenzofurans and dioxin-like polychlorinated biphenyls is up to 0.3pg/g.

In this still further embodiment, more preferably (a) the total amountof polyaromatic hydrocarbons in the composition is up to 0.05 μg/kg,(b), the total amount of dioxins, furans, dibenzeno-para-dioxins andpolychlorinated dibenzofurans is up to 0.2 pg/g, and (d) the totalamount of dioxins, furans, dibenzeno-para-dioxins and polychlorinateddibenzofurans and dioxin-like polychlorinated biphenyls is up to 0.3pg/g.

Typically, in this still further embodiment, the content of isomericimpurities is up to 1.5 wt %. Typically, the content of isomericimpurities is up to 1 wt %, preferably up to 0.5 wt %, more preferablyup to 0.25 wt %, even more preferably up to 0.25 wt %, and mostpreferably up to 0.1 wt %.

The inventors have also found that oils of high purity can be producedwhich avoid the problems of isomerization, peroxidation andoligomerization associated with distilled oils. The quantity of isomericimpurities present in a PUFA product of the present invention willdepend on the amount of isomeric impurities present in the feed mixture.Crucially, though, the amount of isomeric impurities is not increased bythe process of the present invention, unlike distillation. Thus, thelimit of the content of isomers in the PUFA product is the isomericcontent of the starting material. If the starting material has noisomers present, then the resultant PUFA product will also besubstantially free of isomers. This advantage is not observed indistillation.

Thus, in one embodiment, the chromatographic separation process of thepresent invention does not substantially increase the amount of isomericimpurities in the PUFA product relative to the amount of isomericimpurities present in the feed mixture. “Substantially increase” istypically understood to mean increase by 10 wt % or less, preferably 5wt % or less, more preferably 3 wt % or less, even more preferably 1 wt% or less, yet more preferably 0.5 wt % or less, and most preferably 0.1wt % or less.

Thus, in a yet further embodiment, the present invention also provides acomposition comprising a PUFA product, wherein the content of isomericimpurities is up to 1.5 wt %. Typically, the composition contains anamount of isomeric impurities up to this amount. Typically, the contentof isomeric impurities is up to 1 wt %, preferably up to 0.5 wt %, morepreferably up to 0.25 wt %, and most preferably up to 0.1 wt %.Isomerisation is particularly problematic in the preparation of highpurity DHA by distillation, due to the higher temperatures required forseparation. Typically, the PUFA product is DHA, optionally in the formof its ethyl ester. Typically the composition comprises greater than 85wt % PUFA product, preferably greater than 90 wt %, more preferablygreater than 92.5 wt %, most preferably greater than 95 wt %.Preferably, the composition contain comprises greater than 85 wt % DHA,optionally in the form of its ethyl ester, preferably greater than 90 wt%, more preferably greater than 92.5 wt %, most preferably greater than95 wt %. In this embodiment, the composition typically comprises as PUFAproduct DHA, optionally in the form of its ethyl ester, in an amountgreater than 95 wt %, wherein the content of isomeric impurities is upto 1 wt %, preferably up to 0.5 wt %, more preferably up to 0.25 wt %,and most preferably up to 0.1 wt %

The improved process of the invention allows much higher purities ofPUFA product to be achieved efficiently, as both the more and less polarimpurities can be removed in a single process.

The PUFA product of the present invention typically has a purity ofgreater than 80 weight %, preferably greater than 85 weight %, morepreferably greater than 90 weight %, even more preferably greater than95 weight %, yet more preferably greater than 97 weight %, and mostpreferably greater than 99 weight %. When the PUFA product is a singlePUFA or derivative thereof, the concentrations above refer to theconcentration of that PUFA or derivative. When the PUFA product is amixture of two or more PUFAs or derivatives thereof, for example two,the concentrations above refer to the combined concentration of thePUFAs, or derivatives thereof.

The method of the present invention also avoids the problems ofisomerization, peroxidation and oligomerization associated withdistilled oils. The PUFA product of the present invention typically hasa content of isomeric impurities of less than 5 weight %, preferablyless than 3 weight %, and more preferably less than 1 weight %. Asmentioned above, the isomeric impurities include PUFA isomers,peroxidation and oligomerization products. PUFA isomers includepositional and/or geometric isomers. Examples of positional and/orgeometric isomers of EPA include 17E-EPA, 5E-EPA, 5E,8E-EPA, 8E,11E-EPA,5E,14E-EPA, and 5E,8E,11E,17E-EPA. Such isomers are discussed in moredetail in Wijesundera, R. C., et al, Journal of the American OilChemists Society, 1989, vol. 66, no. 12, 1822-1830, the entirety ofwhich is incorporated herein by reference.

In practice, the process of the present invention will generally becontrolled by a computer. The present invention therefore also providesa computer program for controlling a chromatographic apparatus asdefined herein, the computer program containing code means that whenexecuted instruct the apparatus to carry out the process of theinvention.

The following Examples illustrate the invention.

EXAMPLES Example 1

A fish oil derived feedstock (55 weight % EPA EE, 5 weight % DHA EE) isfractionated using an actual moving bed chromatography system usingbonded C18 silica gel (particle size 300 μm) as stationary phase andaqueous methanol as eluent according to the system schematicallyillustrated in FIG. 8. 15 columns (diameter: 76.29 mm, length: 914.40mm) are connected in series as shown in FIG. 8.

The operating parameters and flowrates are as follows for eightdifferent cases. For the conditions below, EPA EE is produced at a highlevel of purity (85 to 98% by GC FAMES). GC FAMES traces of the zone 1extract and raffinate, and of the zone 2 extract and raffinate are shownas FIGS. 11 and 12 respectively.

Example 1a

Step time: 750 secsCycle time: 200 minsFeedstock (F) feed rate: 70 ml/minDesorbent (D) feed rate: 850 ml/minExtract rate: 425 ml/minRaffinate rate: 495 ml/min

Example 1b

Step time: 250 secsCycle time: 66.67 minsFeedstock (F) feed rate: 210 ml/minDesorbent (D) feed rate: 2550 ml/minExtract rate: 1275 ml/minRaffinate rate: 1485 ml/min

Example 1c

Step time: 500 secsCycle time: 133.33 minsFeedstock (F) feed rate: 25 ml/minDesorbent feed rate (D1) in first zone: 2050 ml/minExtract container accumulation rate (E1) in first zone: 1125 ml/minExtract recycle rate (D1-E1) in first zone: 925 ml/minRaffinate rate (R1) in first zone: 950 ml/minDesorbent feed rate (D2) in second zone: 1700 ml/minExtract container accumulation rate (E2) in second zone: 900 ml/minExtract recycle rate (D2-E2) in second zone: 800 ml/minRaffinate rate (R2) in second zone: 800 ml/min

Example 1d

Step time: 250 secsCycle time: 66.67 minsFeedstock (F) feed rate: 50 ml/minDesorbent feed rate (D1) in first zone: 4125 ml/minExtract container accumulation rate (E1) in first zone: 2250 ml/minExtract recycle rate (D1-E1) in first zone: 1875 ml/minRaffinate rate (R1) in first zone: 1925 ml/minDesorbent feed rate (D2) in second zone: 3375 ml/minExtract container accumulation rate (E2) in second zone: 1800 ml/minExtract recycle rate (D2-E2) in second zone: 1575 ml/minRaffinate rate (R2) in second zone: 1575 ml/min

Example 1e

Step time: 500 secsCycle time: 133.33 minsFeedstock (F) feed rate: 50 ml/minDesorbent feed rate (D1) in first zone: 4000 ml/minExtract container accumulation rate (E1) in first zone: 2250 ml/minExtract recycle rate (D1-E1) in first zone: 1750 ml/minRaffinate rate (R1) in first zone: 1800 ml/minDesorbent feed rate (D2) in second zone: 3200 ml/minNet extract accumulation rate (E2) in second zone: 1750 ml/minExtract recycle rate (D2-E2) in second zone: 1450 ml/minRaffinate rate (R2) in second zone: 1450 ml/min

Example 1f

Step time: 250 secsCycle time: 66.67 minsFeedstock (F) feed rate: 100 ml/minDesorbent feed rate (D1) in first zone: 4050 ml/minExtract container accumulation rate (E1) in first zone: 2100 ml/minExtract recycle rate (D1-E1) in first zone: 1950 ml/minRaffinate rate (R1) in first zone: 2050 ml/minDesorbent feed rate (D2) in second zone: 3300 ml/minNet extract accumulation rate (E2) in second zone: 1700 ml/minExtract recycle rate (D2-E2) in second zone: 1600 ml/minRaffinate rate (R2) in second zone: 1600 ml/min

Example 1g

Step time: 500 secsCycle time: 133.33 minsFeedstock (F) feed rate: 25 ml/minDesorbent feed rate (D1) in first zone: 1275 ml/minExtract container accumulation rate (E1) in first zone: 750 ml/minExtract recycle rate (D1-E1) in first zone: 550 ml/minRaffinate rate (R1) in first zone: 575 ml/minDesorbent feed rate (D2) in second zone: 1275 ml/minNet extract accumulation rate (E2) in second zone: 950 ml/minExtract recycle rate (D2-E2) in second zone: 325 ml/minRaffinate rate (R2) in second zone: 325 ml/min

Example 1h

Step time: 250 secsCycle time: 66.67 minsFeedstock (F) feed rate: 50 ml/minDesorbent feed rate (D1) in first zone: 2550 ml/minExtract container accumulation rate (E1) in first zone: 1500 ml/minExtract recycle rate (D1-E1) in first zone: 950 ml/minRaffinate rate (R1) in first zone: 1000 ml/minDesorbent feed rate (D2) in second zone: 2000 ml/minNet extract accumulation rate (E2) in second zone: 900 ml/minExtract recycle rate (D2-E2) in second zone: 600 ml/minRaffinate rate (R2) in second zone: 600 ml/min

Example 2

A fish oil derived feedstock comprising eicosatetraenoic acid ethylester (ETA EE), EPA EE, isomers thereof and DHA EE was fractionatedusing an actual moving bed chromatography system using bonded C18 silicagel (particle size 40-60 μm) as stationary phase and aqueous methanol aseluent according to the system schematically illustrated in FIG. 10. 19columns (diameter: 10 mm, length: 250 mm) are connected in series asshown in FIG. 10.

The operating parameters and flowrates are as follows.

Cycle time: 600 secsFeedstock (F) feed rate: 0.5 ml/minDesorbent (D1, 100% methanol) feed rate into the first zone: 6 ml/minDesorbent (D2, 99% methanol/1% water) feed rate into the first zone: 6ml/minExtract (E1′) rate from the first zone: 3 ml/minExtract (E1) rate from the first zone: 1.9 ml/minRaffinate (R1) rate from the first zone: 4.6 ml/minExtract (E2) rate from the second zone: 2.4 ml/minRaffinate (R2) rate from the second zone: 4.6 ml/min

Again, EPA EE was produced at a high level of purity (greater than 90weight %, greater than 95 weight %, greater than 98 weight %).

Example 3

A fish oil derived feedstock (55 weight % EPA EE, 5 weight % DHA EE) wasfractionated using an actual moving bed chromatography system usingbonded C18 silica gel (particle size 300 μm, particle porosity 150angstroms) as stationary phase and aqueous methanol as eluent accordingto the system schematically illustrated in FIG. 8. 15 columns (diameter:10 mm, length: 250 mm) are connected in series as shown in FIG. 8.

The operating parameters and flowrates are as follows.

Cycle time: 380 secsFeedstock (F) feed rate: 0.5 ml/minDesorbent (D, 98.5% methanol/1.5% water) feed rate into the first zone:9 mUminWater rich phase (W; 85% methanol/15% water) feed rate into the firstzone: 3.1 ml/minExtract (E1) rate from the first zone: 4 ml/minRaffinate (R1) rate from the first zone: 8.6 ml/minDesorbent (D, 97% methanol/3% water) feed rate into the second zone:10.8 ml/minWater rich phase (W, 85% methanol/15% water) feed rate into the secondzone: 3.1 ml/minExtract (E2) rate from the second zone: 4.1 ml/minRaffinate (R2) rate from the second zone: 10.3 ml/min

EPA EE was produced at a high level of purity (>95% purity). A GC traceof the product is shown as FIG. 13

Example 4

A fish oil derived feedstock (70 weight % DHA EE, 7 weight % EPA EE) isfractionated using an actual moving bed chromatography system usingbonded C18 silica gel (particle size 300 μm) as stationary phase andaqueous methanol as eluent according to the system schematicallyillustrated in FIG. 8. 15 columns (diameter: 76.29 mm, length: 914.40mm) are connected in series as shown in FIG. 8.

The operating parameters and flowrates are as follows.

Step time: 600 secsCycle time: 160 minsFeedstock (F) feed rate: 25 ml/minDesorbent feed rate (D1) in first zone: 2062.5 ml/minExtract rate (E1) in first zone: 900 ml/minRaffinate rate (R1) in first zone: 1187.5 ml/minDesorbent feed rate (D2) in second zone: 1500 ml/minExtract rate (E2) in second zone: 450 ml/minRaffinate rate (R2) in second zone: 1050 ml/min

DHA EE is produced at a high level of purity (>97% by GC FAMES). A GCFAMES trace of the zone 2 extract is shown as FIG. 14.

Example 5

A fish oil derived feedstock (33 weight % EPA EE, 22 weight % DHA EE) isfractionated using an actual moving bed chromatography system usingbonded C18 silica gel (particle size 300 μm) as stationary phase andaqueous methanol as eluent according to the system schematicallyillustrated in FIG. 8. 15 columns (diameter: 76.29 mm, length: 914.40mm) are connected in series as shown in FIG. 8.

The operating parameters and flowrates are as follows.

Step time: 380 secsCycle time: 101.33 minsFeedstock (F) feed rate: 40 ml/minDesorbent feed rate (D1) in first zone: 1950 ml/minExtract rate (E1) in first zone: 825 ml/minRaffinate rate (R1) in first zone: 1165 ml/minDesorbent feed rate (D2) in second zone: 1425 ml/minExtract rate (E2) in second zone: 787.5 ml/minRaffinate rate (R2) in second zone: 637.5 ml/min

A mixture of EPA EE and DHA EE is produced at a high level of purity(>80% total EPA EE and DHA EE).

Example 6

An experiment was carried out to compare the amount of environmentalpollutants present in two PUFA products according to the presentinvention with similar oils prepared by distillation. The pollutantprofiles of the oils are shown in Table 1 below.

TABLE 1 PUFA product PUFA product according to according to ReleaseDistilled Distilled the invention the invention Parameter Specificationoil [1] oil [2] [1] [2] Polyaromatic Hydrocarbons (PAH) (μg/kg)Benzo(a)pyrene NMT 2.0 0.90 0.90 <0.05 <0.05 Impurities Dioxins andFurans PCDDs and NMT 2.0 0.46 0.37 0.2 0.184 PCDFs¹⁾ (pgWHO-PCDD/F-TEQ/g) PCBs (mg/kg) NMT 0.09 0.0037 0.0103 0.0007 0.0012 Sumof Dioxins, Furans and Dioxin- NMT 10.0 1.03 0.466 0.30 0.298 likePCBs²⁾ (pg WHO- PCDD/F-PCB-TEQ/g) ¹⁾Dioxin limits include the sum ofpolychlorinated dibenzeno-para-dioxins (PCDDs) and polychlorinateddibenzofurans (PCDFs) and expressed in World Health Organisation (WHO)toxic equivalents using WHO-toxic equivalent factors (TEFs). This meansthat analytical results relating to 17 individual dioxin congeners oftoxicological concern are expressed in a single quantifiable unit: TCDDtoxic equivalent concentration or TEQ ²⁾Maximum for dioxin and Furansremains at 2 pg/g

Example 7

An experiment was carried out to determine the amount of isomericimpurities present in an oil prepared according to the present inventioncompared with an equivalent oil prepared by distillation.

A GC trace of the DHA-rich oil prepared in accordance with the inventionis shown as FIG. 14. There is no evidence of isomeric impurities in theGC trace.

A GC trace of the oil prepared by distillation is shown as FIG. 15. Thefour peaks with longer elution times than the DHA peak correspond to DHAisomers. From the GC trace it can be seen that the oil prepared bydistillation contains about 1.5 wt % of isomeric impurities.

Example 8

Two EPA-rich products of the process of the present invention werecompared with EPA-rich oils produced by distillation. The wt % analysisof their component PUFAs is shown below.

PUFA product PUFA product according to according to Dis- Dis- theinvention the invention tilled tilled Fatty Acid [1] [2] oil [1] oil [2]EPA (C20:5n-3) 98.33  97.04  98.09  98.14  DHA (C22:6n-3) 0.15 <LOD 0.34<LOD C18:3 n-3 <LOD 0.28 0.24 <LOD C18:4 n-3 0.33 0.20 0.14 0.26 C20:4n-3 0.14 0.45 0.18 0.46 C21:5 n-3 <LOD <LOD <LOD <LOD C22:5 n-3 0.32<LOD <LOD <LOD Total Omega-3 99.27  97.97  98.94  98.86  C18:3n-6 <LOD<LOD 0.05 <LOD C20:3 n-6 <LOD <LOD 0.13 0.11 C20:4 n-6 <LOD 0.21 0.260.37 Total Omega-6 <LOD 0.21 0.44 0.48

Example 9

An EPA/DHA-rich product of the process of the present invention wascompared with an EPA/DHA-rich oil produced by distillation. The wt %analysis of their component PUFAs is shown below.

Maxomega Ethyl Ester Distilled Ethyl Ester (Omega-3 90 ethyl (Omega-3 90ethyl esters) esters¹) Fatty Acid Area % Area % EPA (C20:5n-3) 53.3 46.6DHA (C22:6n-3) 32.9 38.2 TOTAL EPA + 86.2 84.8 DHA C18:3 n-3 0.3 0.1C18:4 n-3 1.2 2.0 C20:4 n-3 1.8 0.6 C21:5 n-3 2.7 1.8 C22:5 n-3 5.0 3.8Total Omega-3 97.2 93.1 C18:2 n-6 0.2 0.1 C18:3n-6 <0.1 0.2 C20:3 n-6<0.1 0.1 C20:4 n-6 2.0 2.6 C22:4 n-6 <0.1 0.1 C22:5 n-6 0.6 1.0 TotalOmega-6 2.8 4.1

What is claimed is:
 1. A composition comprising a PUFA product, whereinthe PUFA product is EPA present in an amount greater than 93 wt %,wherein the total content of ω-6 polyunsaturated fatty acids is up to0.40 wt %, and wherein the total content of eicosatetraenoic acid is upto 1 wt %.
 2. The composition according to claim 1, wherein the totalcontent of eicosatetraenoic acid is 0.05 wt % or greater.
 3. Thecomposition according to claim 1, wherein the composition (a) comprisesgreater than 96.5 wt % EPA, up to 1 wt % DHA, up to 1 wt % α-linolenicacid, up to 1 wt % stearidonic acid, up to 1 wt % eicosatetraenoic acid,up to 1 wt % docosapentaenoic acid, and up to 0.25 wt % arachidonicacid; or (b) comprises greater than 96.5 wt % EPA, up to 0.2 wt % DHA,up to 0.3 wt % α-linolenic acid, up to 0.4 wt % stearidonic acid, up to0.5 wt % eicosatetraenoic acid, up to 0.35 wt % docosapentaenoic acid,and up to 0.25 wt % arachidonic acid.
 4. The composition according toclaim 1, wherein the composition (a) comprises from 96.5 to 99 wt % EPA,up to 0.6 wt % DHA, up to 0.6 wt % α-linolenic acid, from 0.15 to 0.6 wt% stearidonic acid, from 0.1 to 0.75 wt % eicosatetraenoic acid, up to0.6 wt % docosapentaenoic acid, and up to 0.6 wt % arachidonic acid; or(b) comprises from 96.5 to 99 wt % EPA, up to 0.2 wt % DHA, up to 0.3 wt% α-linolenic acid, from 0.15 to 0.4 wt % stearidonic acid, from 0.1 to0.5 wt % eicosatetraenoic acid, up to 0.35 wt % docosapentaenoic acid,and up to 0.25 wt % arachidonic acid.
 5. The composition according toclaim 1, wherein the composition (a) comprises from 98 to 99 wt % EPA,from 0.1 to 0.3 wt % DHA, from 0.3 to 0.35 wt % stearidonic acid, from0.1 to 0.3 wt % eicosatetraenoic acid, and from 0.3 to 0.35 wt %docosapentaenoic acid; or (b) comprises from 96.5 to 99 wt % EPA, from0.1 to 0.5 wt % DHA, from 0.1 to 0.5 wt % stearidonic acid, from 0.1 to0.5 wt % eicosatetraenoic acid, from 0.1 to 0.5 wt % docosapentaenoicacid, and from 0.1 to 0.3 wt % arachidonic acid; or (c) comprises from98 to 99 wt % EPA, from 0.1 to 0.2 wt % DHA, from 0.3 to 0.35 wt %stearidonic acid, from 0.1 to 0.2 wt % eicosatetraenoic acid, and from0.3 to 0.35 wt % docosapentaenoic acid; or (d) comprises from 96.5 to97.5 wt % EPA, from 0.25 to 0.35 wt % α-linolenic acid, from 0.18 to0.24 wt % stearidonic acid, from 0.4 to 0.46 wt % eicosatetraenoic acid,and from 0.15 to 0.25 wt % arachidonic acid.
 6. A composition comprisinga PUFA product, wherein the PUFA product is a mixture of EPA and DHA,wherein (i) the total content of EPA and DHA is 80 wt % or greater, (ii)the content of EPA is from 41 to 60 wt % and the content of DHA is from16 to 48 wt %, and (iii) the total content of ω-3 polyunsaturated fattyacids is 94 wt % or greater and/or the total content of ω-6polyunsaturated fatty acids is up to 4 wt %; and wherein (a) the totalcontent of ω-3 polyunsaturated fatty acids is 97 wt % or greater, and/or(b) the total content of stearidonic acid is up to 1.5 wt %, and/or (c)the total content of docosapentaenoic acid is from 4 to 5.25 wt %,and/or (d) the total content of adrenic acid is up to 0.1 wt %.
 7. Thecomposition according to claim 6, wherein the total content ofstearidonic acid is from 0.05 wt % to 1.5 wt %.
 8. The compositionaccording to claim 6, wherein the total content of adrenic acid is from0.05 wt % to 0.1 wt %.
 9. The composition according to claim 6, whereinthe composition comprises from 50 to 55 wt % EPA, from 30 to 35 wt %DHA, up to 0.4 wt % α-linolenic acid, up to 1.25 wt % stearidonic acid,up to 1.9 wt % eicosatetraenoic acid, up to 2.75 wt %heneicosapentaenoic acid, up to 5.25 wt % docosapentaenoic acid, up to0.25 wt % linoleic acid, up to 0.1 wt % gamma-linolenic acid, up to 0.1wt % dihommo-gamma-linolenic acid, up to 2.1 wt % arachidonic acid, upto 0.1 wt % adrenic acid, and up to 0.75 wt % docosapentaenoic (ω-6)acid.
 10. The composition according to claim 6, wherein the compositioncomprises from 50 to 55 wt % EPA, from 30 to 35 wt % DHA, from 0.2 to0.4 wt % α-linolenic acid, up to 1.25 wt % stearidonic acid, from 1.0 to1.9 wt % eicosatetraenoic acid, from 2 to 2.75 wt % heneicosapentaenoicacid, from 4 to 5.25 wt % docosapentaenoic acid, from 0.15 to 0.25 wt %linoleic acid, up to 0.1 wt % gamma-linolenic acid, up to 0.1 wt %dihommo-gamma-linolenic acid, up to 2.1 wt % arachidonic acid, up to 0.1wt % adrenic acid, and up to 0.75 wt % docosapentaenoic (ω-6) acid. 11.The composition according to claim 1, wherein (a) a total amount ofpolyaromatic hydrocarbons in the composition is up to 0.89 μg/kg, (b) atotal amount of dioxins, furans, dibenzeno-para-dioxins andpolychlorinated dibenzofurans is up to 0.35 pg/g, (c) a total amount ofpolychlorinated biphenyls is up to 0.0035 mg/kg, and/or (d) a totalamount of dioxins, furans, dibenzeno-para-dioxins, polychlorinateddibenzofurans and dioxin-like polychlorinated biphenyls is up to 1 pg/g.12. The composition according to claim 1, wherein a content of isomericimpurities is up to 1.5 wt %.
 13. The composition according to claim 12,wherein the content of isomeric impurities is up to 1 wt %, up to 0.5 wt%, up to 0.25 wt %, or up to 0.1 wt %.
 14. The composition according toclaim 7, wherein (a) a total amount of polyaromatic hydrocarbons in thecomposition is up to 0.89 μg/kg, (b) a total amount of dioxins, furans,dibenzeno-para-dioxins and polychlorinated dibenzofurans is up to 0.35pg/g, (c) a total amount of polychlorinated biphenyls is up to 0.0035mg/kg, and/or (d) a total amount of dioxins, furans,dibenzeno-para-dioxins, polychlorinated dibenzofurans and dioxin-likepolychlorinated biphenyls is up to 1 pg/g.
 15. The composition accordingto claim 6, wherein a content of isomeric impurities is up to 1.5 wt %.16. The composition according to claim 15, wherein the content ofisomeric impurities is up to 1 wt %, up to 0.5 wt %, up to 0.25 wt %, orup to 0.1 wt %.
 17. A composition comprising a PUFA product, wherein (a)a total amount of polyaromatic hydrocarbons in the composition is up to0.89 μg/kg, (b), a total amount of dioxins, furans,dibenzeno-para-dioxins and polychlorinated dibenzofurans is up to 0.35pg/g (c) a total amount of polychlorinated biphenyls is up to 0.0035mg/kg, and/or (d) a total amount of dioxins, furans,dibenzeno-para-dioxins, polychlorinated dibenzofurans and dioxin-likepolychlorinated biphenyls is up to 1 pg/g.
 18. The composition accordingto claim 17, wherein the PUFA product is DHA, and the compositioncomprises greater than 85 wt % PUFA product.
 19. A compositioncomprising a PUFA product, wherein the content of isomeric impurities isup to 1.5 wt %.
 20. The composition according to claim 19, wherein thecontent of isomeric impurities is up to 1 wt %, up to 0.5 wt %, up to0.25 wt %, or up to 0.1 wt %.
 21. The composition according to claim 19,wherein the PUFA product is DHA, and the composition comprises greaterthan 85 wt % PUFA product.